Lack of lipoprotein-dependent effects on the cytotoxic interactions of Actinobacillus actinomycetemcomitans leukotoxin with human neutrophils.

A high odds ratio has been reported for hyperlipidemia and periodontal diseases in humans, and the severity of periodontitis seems to correlate with the hyperlipidemic status of the patients. Early studies indicated that the lipoprotein-containing fraction of the serum enhances the leukotoxic activity of the periodontopathogen Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL). The protease inhibitors of normal serum account for this enhancement, while delipidated serum has no effect on the leukotoxin-dependent PMNL cytolysis. No information exists for the effect of serum lipoproteins or hyperlipidemic serum. The aim of this study was to evaluate the role of serum lipoproteins in the interaction of the leukotoxin of A. actinomycetemcomitans with human PMNL. Purified leukotoxin was mixed with human PMNL prepared from venous blood of healthy subjects and various varying amounts of hyperlipidemic or delipidated serum, or purified serum lipoproteins. The cytolytic activity of leukotoxin was determined by activity of the cytosol enzyme lactate dehydrogenase released from injured PMNL. The degranulating activity of the toxin was measured through the release of the granule components elastase and lactoferrin. Normal human serum without leukotoxin-neutralizing antibodies caused a 4-fold enhancement of the leukotoxic activity when present at concentrations of 5-10% in the reaction mixture. Serum lipoproteins had no effect when added at concentrations that occur normally in serum. At high concentrations, purified low density and very low-density lipoproteins increased the leukotoxicity of the mixture. Nevertheless, hyperlipidemic serum prepared from a normal serum by the addition of autologous lipoproteins had no influence on the leukotoxin-caused cytolysis compared to the normal serum. Pre-incubation of PMNL for 1 h in hyperlipidemic or delipidated serum had no effect on the leukotoxin-induced degranulation of PMNL. The results indicate that the cytotoxic interactions of A. actinomycetemcomitans leukotoxin against human PMNL are not influenced by the presence of serum lipoproteins.

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

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.

Effects of the heparin-mimicking compound RG-13577 on lipoprotein lipase and on lipase mediated binding of LDL to cells.

Lipoprotein lipase (LPL) has high affinity for heparin and heparin-like compounds. In vivo the enzyme is attached to heparan sulfate proteoglycans on the endothelium of capillaries and larger blood vessels. The enzyme is released from these sites after intravenous injection of heparin. One has here investigated the effects of RG-13577 on LPL, both after intravenous injection to rats and under cell culture conditions. RG-13577 is a heparin-mimicking compound known to prevent angiogenesis by interference with binding of growth factors to cells. It has therefore been considered for use in cancer therapy as well as for prevention of atherosclerosis and restenosis. It was found that intravenously injected RG-13577 released both LPL and hepatic lipase (HL) to the blood. Binding of LPL in extrahepatic tissues was prevented and clearance of radiolabeled LPL from the circulation was delayed. Furthermore, RG-13577 released LPL from extracellular matrix (ECM) produced by endothelial cells and from THP-1 monocyte-derived macrophages. Lipase-mediated binding and uptake of human LDL in these cells was also prevented by RG-13577. Thus, in the test systems RG-13577 had the same effects as heparin, but on a molar basis RG-13577 was in all cases less effective.

Binding of low density lipoproteins to lipoprotein lipase is dependent on lipids but not on apolipoprotein B.

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.

Mild oxidation of lipoproteins increases their affinity for surfaces covered by heparan sulfate and lipoprotein lipase.

Lipoprotein lipase (LPL) is present in cells involved in development of atherosclerosis (endothelial cells, smooth muscle cells, and macrophages). A direct involvement of LPL in atherogenesis has been suggested. Previously we used the surface plasmon resonance technique to study the interaction of lipoproteins with surfaces covered by heparan sulfate proteoglycans (HSPG) and LPL [A. Lookene et al. (1997) Biochemistry 36, 5267-5275]. The binding was much increased by the presence of LPL. Here we demonstrate that mild oxidation of low-density-lipoprotein (LDL) and very-low-density lipoprotein (VLDL) in vitro increases their binding to surfaces covered by HSPG and LPL, while extensive oxidation decreases it. Similar results were obtained with a lipid emulsion (Intralipid), indicating that oxidation-induced changes of the lipid part could explain the effects. LPL increased binding and uptake of the mildly oxidized (compared to nonoxidized) LDL by THP-I monocyte-derived macrophages. Our studies indicate that LPL has the highest affinity for mildly oxidized LDL and support its involvement in development of atherosclerosis.

[Does the cholesterol content in blood cells depend on its level in plasma?]. / Zavisit li soderzhanie kholesterina v kletkakh krovi ot ego urovnia v plazme.

The cholesterol content in human erythrocytes, granulocytes and the total leucocyte fraction with varying levels of plasma cholesterol has been studied. There was no correlation between the cell cholesterol content and the levels of total, LDL and HDL cholesterol in the plasma. The mean cholesterol content in one cell has been found to be equal to 1.26 x 10(-13) g in the erythrocytes, 2.08 x 10(-12) g in the granulocytes and 3.35 x 10(-12) g in the total leucocyte fraction.

[Cryohemosorption--a method of removal of lipoprotein-antibody immune complexes and other atherogenic substances in patients with atherosclerosis]. / Krioplazmosorbtsiia--metod udaleniia immunnykh kompleksov lipoproteid-antitelo i drugikh aterogennykh substantsii u bol'nykh aterosklerozom.

The efficacy of extracorporeal cryohemosorption was estimated in the treatment of atherosclerotic patients in terms of the autoimmune theory of the pathogenesis of this disease. There was a slight decrease in the plasma levels of total cholesterol and triglycerides, there was over 2-fold reduction in the plasma levels of fibrinogen and fibronectin. All major components of apo B-containing lipoproteins and immunoglobulin G were found as part of the cryoprecipitate. An extremely high concentration of lipid hydroperoxides in the precipitate suggest that cryoprecipitation removes not only autoimmune complexes, but highly-atherogenic peroxide-modified lipoproteins. The three-fold decrease in the levels of lipoprotein-antibody complexes resulted in lower atherogenicity of apo B-containing lipoproteins. It is suggested that the mechanism responsible for beneficial clinical action of cryohemosorption sessions is largely associated with the removal of autoimmune lipoprotein-antibody complexes and peroxide-modified apo B-containing lipoproteins than correction of lipid metabolism.