Effects ofAggregatibacter actinomycetemcomitansleukotoxin on neutrophil migration and extracellular trap formation.
BACKGROUND
Aggressive periodontitis is associated with the presence ofAggregatibacter actinomycetemcomitans, a leukotoxin (Ltx)-producing periodontal pathogen. Ltx has the ability to lyse white blood cells including neutrophils.


OBJECTIVE
This study was aimed at investigating the interactions between neutrophils and Ltx with regard to the chemotactic properties of Ltx and the release of neutrophil extracellular traps (NETs).


METHODS
Neutrophils from healthy blood donors were isolated and incubated for 30 min and 3 h with increasing concentrations of Ltx (1, 10, and 100 ng/mL) as well as withA. actinomycetemcomitansstrains (NCTC 9710 and HK 1651) producing different levels of Ltx. Formation of NETs and cell lysis were assessed by microscopy, fluorescence-based assays, and measurement of released lactate dehydrogenase. Neutrophil migration in response to different Ltx gradients was monitored by real-time video microscopy, and image analysis was performed using ImageJ software.


RESULTS
Although Ltx (10 and 100 ng/mL) and the leukotoxicA. actinomycetemcomitansstrain HK 1651 lysed some neutrophils, other cells were still capable of performing NETosis in a concentration-dependent manner. Low doses of Ltx and the weakly leukotoxic strain NCTC 9710 did not lead to neutrophil lysis, but did induce some NETosis. Furthermore, all three concentrations of Ltx enhanced random neutrophil movement; however, low directional accuracy was observed compared with the positive control (fMLP).


CONCLUSIONS
The results indicate that Ltx acts both as a neutrophil activator and also causes cell death. In addition, Ltx directly induces NETosis in neutrophils prior to cell lysis. In future studies, the underlying pathways involved in Ltx-meditated neutrophil activation and NETosis need to be investigated further.
Periodontal Research Group, Birmingham Dental School & Hospital, Birmingham, United Kingdom.|Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Bonn, Germany; j.hirschfeld@bham.ac.uk.
Josefine
J
Hirschfeld
Periodontal Research Group, Birmingham Dental School & Hospital, Birmingham, United Kingdom.
Helen M
HM
Roberts
Periodontal Research Group, Birmingham Dental School & Hospital, Birmingham, United Kingdom.
Iain L C
IL
Chapple
Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Bonn, Germany.
Marijo
M
Parčina
Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Bonn, Germany.
Søren
S
Jepsen
Section Molecular Periodontology, Department of Odontology, Umeå University, Umeå, Sweden.
Anders
A
Johansson
Section Oral Microbiology, Department of Odontology, Umeå University, Umeå, Sweden.
Rolf
R
Claesson
Aggregatibacter actinomycetemcomitans; NETs; aggressive periodontitis; chemotaxis; leukotoxin; migration; neutrophils