title: Protease inhibitors, the responsible components for the serum-dependent enhancement of Actinobacillus actinomycetemcomitans leukotoxicity.
abstract: Serum enhances the leukotoxic activity of Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL) by a mechanism that still is unknown. Early attempts to identify the serum components responsible for this enhancement gave no conclusive results, but indicated that the lipoprotein-containing fraction of the serum was involved in the interaction. This study aimed to clarify the role of serum lipoproteins in the leukotoxin 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 peripheral blood and incubated with purified leukotoxin in the presence of serum or purified serum components or lipoprotein-deficient serum. Leukotoxin was also incubated with purified elastase and cathepsin G or with enzyme mixtures from degranulated PMNL. The leukotoxic activity in these mixtures was determined as the extracellular release of lactate dehydrogenase from PMNL. Cleavage of the toxin was showed by gel electrophoresis and Western blot. Morphological changes in PMNL from the above mixtures were examined by electron microscopy. Enzymes from degranulated PMNL cleaved leukotoxin to non-cytotoxic 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 alpha2-macroglobulin and alpha1-proteinase inhibitor. Under these conditions enhanced PMNL lysis was also observed. A similar enhancement of PMNL lysis was found when PMNL degranulation was blocked by EDTA. On the other hand, lipoprotein-deficient serum had no influence on the leukotoxic activity. The results indicate that the increased leukotoxicity of A. actinomycetemcomitans observed in the presence of human serum is caused by the serum protease inhibitors that counteract proteolytic degradation of leukotoxin. The degradation is caused by enzymes from degranulated PMNL triggered by leukotoxin.
authors.affiliation: Department of Odontology, Umeå University, Sweden. anders.johansson@odont.umu.se
authors.forename: A
authors.initials: A
authors.lastname: Johansson
authors.forename: R
authors.initials: R
authors.lastname: Claesson
authors.forename: G
authors.initials: G
authors.lastname: Belibasakis
authors.forename: E
authors.initials: E
authors.lastname: Makoveichuk
authors.forename: L
authors.initials: L
authors.lastname: Hänström
authors.forename: G
authors.initials: G
authors.lastname: Olivecrona
authors.forename: G
authors.initials: G
authors.lastname: Sandström
authors.forename: S
authors.initials: S
authors.lastname: Kalfas
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000378:metabolism* / Q000502:physiology; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D016923:Cell Death; D015550:Cell Degranulation / Q000187:drug effects / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D003603:Cytotoxins / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005098:Exotoxins / Q000378:metabolism*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008074:Lipoproteins / Q000097:blood; D008854:Microscopy, Electron; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000382:microbiology; D010196:Pancreatic Elastase / Q000378:metabolism; D011480:Protease Inhibitors / Q000097:blood*; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D002614:Chelating Agents; D003603:Cytotoxins; D005098:Exotoxins; D008074:Lipoproteins; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; D000511:alpha-Macroglobulins; C018579:leukotoxin; D004492:Edetic Acid; D007770:L-Lactate Dehydrogenase; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase