Protease inhibitors, the responsible components for the serum-dependent enhancement of Actinobacillus actinomycetemcomitans leukotoxicity

Serum enhances the leukotoxic activity of Actinobacillus actinomycetemcomit ans against human polymorphonuclear leukocytes (PMNL) by a mechanism that s till is unknown. Early attempts to identify the serum components responsibl e for this enhancement gave no conclusive results, but indicated that the l ipoprotein-containing fraction of the serum was involved in the interaction . This study aimed to clarify the role of serum lipoproteins in the leukoto xin interaction, and to identify other serum components involved. The main hypothesis examined was that the leukotoxicity enhancement might depend on serum protease inhibitors that block proteolytic cleavage of leukotoxin by enzymes released from the leukocytes. PMNL were isolated from human periphe ral blood and incubated with purified leukotoxin in the presence of serum o r purified serum components or lipoprotein-deficient serum. Leukotoxin was also incubated with purified elastase and cathepsin G or with enzyme mixtur es from degranulated PMNL. The leukotoxic activity in these mixtures was de termined as the extracellular release of lactate dehydrogenase from PMNL. C leavage of the toxin was showed by gel electrophoresis and Western blot. Mo rphological changes in PMNL from the above mixtures were examined by electr on microscopy. Enzymes from degranulated PMNL cleaved leukotoxin to non-cyt otoxic fragments. Elastase and cathepsin G were mainly responsible for the cleavage. Inhibition of leukotoxin degradation was found in the presence of whole serum or of the serum protease inhibitors alpha (2)-macroglobulin an d alpha (1)-proteinase inhibitor. Under these conditions enhanced PMNL lysi s was also observed. A similar enhancement or PMNL lysis was found when PMN L degranulation was blocked by EDTA. On the other hand, lipoprotein-deficie nt serum had no influence on the leukotoxic activity. The results indicate that the increased leukotoxicity of A. actinomycetemcomitans observed in th e presence of human serum is caused by the serum protease inhibitors that c ounteract proteolytic degradation of leukotoxin. The degradation is caused by enzymes from degranulated PMNL triggered by leukotoxin.