Polymorphonuclear leukocyte degranulation induced by leukotoxin from Actinobacillus actinomycetemcomitans

Abstract

The ability of leukotoxin from Actinobacillus actinomycetemcomitans to induce release of lysosomal constituents was studied with human polymorphonuclear leukocytes (PMNL). Leukotoxin purified from A. actinomycetemcomitans or bacterial cells of a leukotoxic strain were mixed with human PMNL and the suspension was incubated under anaerobic conditions. Samples were taken at certain time intervals to examine the cell morphology of PMNL by electron microscopy and the extracellular concentrations of the granule components lactoferrin and elastase by enzyme-linked immunosorbent assay (ELISA). Electron microscopy revealed that within 10 min of exposure to leukotoxin, the number of intracellular granules was markedly reduced and the remaining granules were translocated to the periphery in PMNL. At the same time, the extracellular concentrations of lactoferrin and elastase were elevated, while that of the cytosolic enzyme lactate dehydrogenase, an indicator of cell lysis, remained low. The lysosome molecules CD63 and CD66b were also exposed on the PMNL surface, indicating fusion of lysosomes with the plasma membrane. These effects were completely abolished by the addition of anti-leukotoxin serum. Pre-incubation of PMNL with monoclonal antibodies to CD11a and CD18 that recognize alpha- and beta-chains of the LFA-1 integrin, a leukotoxin receptor on PMNL, inhibited the cytolysis, but not the release of granule components. The present results demonstrate the ability of A. actinomycetemcomitans leukotoxin to trigger a rapid release of lysosomal compounds in human PMNL. The release is due to an active process stimulated by the interaction of PMNL with the toxin or toxin-carrying bacteria.

Leukotoxin (Leukothera®) Targets Active Leukocyte Function Antigen-1 (LFA-1) Protein and Triggers a Lysosomal Mediated Cell Death Pathway

“…Johansson et al (25) reported that cells underwent degranulation after LtxA treatment; however, our data do not support this claim because LAMP-1 was never detected on the surface of cells. It is important to note that this group examined the effect of LtxA on total primary peripheral blood mononuclear cells rather than a single cell type.…”

“…In contrast, cells that were treated with buffer did not change in appearance or staining properties over time (data not shown). This result could imply a mechanism involving degranulation of the cells, as suggested by Johansson et al (25). Presentation of the lysosomal protein LAMP-1 (CD107a) on the cell surface is characteristic of cellular degranulation (26), but we did not observe cell surface expression of LAMP-1 after LtxA treatment (Fig.…”

Aggregatibacter actinomycetemcomitans leukotoxin (LtxA; Leukothera) induces cofilin dephosphorylation and actin depolymerization during killing of malignant monocytes

“…In 2000, Johansson and colleagues (Johansson et al, 2000) reported that LtxA induces rapid degranulation of polymorphonuclear leukocytes prior to lysis of cells. Degranulation is a process that requires actin polymerization.…”

Virulence Mechanisms of Leukotoxin from Aggregatibacter actinomycetemcomitans

“…This property of leukotoxin further supports the importance of electrostatic forces for its association to the bacterial outer membrane. The secreted leukotoxin has been shown to be easily inactivated by environmental proteases and superoxide radicals (Johansson et al, 2000b(Johansson et al, , 2001Balashova et al, 2007). This degradation of the toxin molecule can be inhibited by the presence of superoxide dismutase (SOD) produced by A. actinomycetemcomitans and the naturally occurring protease inhibitors of human serum (Johansson et al, 2001;de Haar et al, 2006;Balashova et al, 2007).…”

“…8). Further analysis of PMNs exposed to leukotoxin showed an extracellular release of proteolytic enzymes from both primary and secondary granules (Johansson 2000b). More over, the interaction between leukotoxin and PMNs mediates activation and release of matrix metalloproteinase 8 (Claesson et al, 2002).…”