Hypersusceptibility of neutrophil granulocytes towards lethal action of free fatty acids contained in enzyme-modified atherogenic low density lipoprotein.

OBJECTIVE: The bulk of LDL entrapped in the arterial intima is modified by hydrolytic enzymes, leading to extensive cleavage of cholesterylesters and liberation of fatty acids. The latter induce apoptosis in endothelial cells but are far less cytotoxic towards macrophages. We have compared the cytotoxic effects of enzymatically modified LDL (E-LDL) on macrophages and polymorphonuclear granulocytes (PMN). METHODS AND RESULTS: E-LDL displayed toxicity towards PMN at far lower concentrations than towards monocyte-derived macrophages. Native or oxidized LDL had no effect. Free fatty acids contained in E-LDL were the cause of the observed toxicity, which could be mimicked by linoleic acid, oleic acid and arachidonic acid. E-LDL provoked Ca(2+) influx and activated PMN, as witnessed by the generation of superoxide anions and peroxidase secretion. Inhibition of either oxidative burst or calcium influx did not diminish the cytotoxicity of E-LDL. Similar to free linoleic acid, E-LDL lysed red blood cells and rapidly rendered cells permeable to propidium iodide. CONCLUSION: Possibly through their capacity to directly perturb cell membranes, free fatty acids contained in E-LDL exert potent cytotoxic effects on PMN. This may be one reason why PMN are not abundantly present in atherosclerotic lesions, and why PMN-depletion suppresses atherogenesis.

Potential protective role of apoprotein J (clusterin) in atherogenesis: binding to enzymatically modified low-density lipoprotein reduces fatty acid-mediated cytotoxicity.

Following entrapment in the arterial intima, low-density lipoprotein (LDL) can be modified by hydrolytic enzymes to yield a lipoprotein derivative that binds C-reactive protein, activates complement, and is rapidly taken up by monocytes/macrophages. Free fatty acids contained in enzymatically modified LDL (E-LDL) render the lipoprotein cytotoxic due to their capacity to trigger programmed cell death. Apoprotein J (ApoJ) alias clusterin is a multifunctional glycoprotein with cytoprotective and anti-inflammatory properties. It interacts with diverse substrates, is present in the intima and the media of arteries with atherosclerotic lesions and is also synthesized by smooth muscle cells during development of atherosclerosis. We report that ApoJ binds to E-LDL but not to native LDL. Binding resulted in marked reduction of cytotoxicity of E-LDL on smooth muscle cells, as revealed by determination of caspase activity, annexin binding, and cellular ATP. ApoJ was detected immunohistochemically in early atherosclerotic lesions, where it was found to co-localize with E-LDL. In atherosclerotic lesions, ApoJ may thus subserve protective functions through its capacity to inactivate C5b-9 complement complexes and by reducing the cytotoxic effects of modified LDL on cells that gain contact with the lipoprotein.

Enzymatically hydrolyzed low-density lipoprotein modulates inflammatory responses in endothelial cells.

There is evidence that low-density lipoprotein (LDL) is modified by hydrolytic enzymes, and that the product (E-LDL) induces selective production of interleukin 8 (IL-8) in endothelial cells. Since nuclear factor-kappaB (NF-kappaB) is a major regulator of IL-8 transcription, we studied its activation in endothelial cells treated with E-LDL. Unexpectedly, the modified lipoprotein not only failed to activate NF-kappaB, but completely blocked its activation by tumour necrosis factor-alpha (TNF-alpha) in EA.hy926-cells, as assessed by electrophoretic mobility shift assays and immunofluorescence. Inhibition occurred upstream of NF-kappaB translocation, as inhibitor of NF-kappaB- (IkappaB)-phosphorylation was suppressed by E-LDL. In contrast to NF-kappaB, transcription factor activator protein-1 (AP-1) proved to be activated. Removal of free fatty acids present in E-LDL obliterated both activation of AP-1 and inhibition of NF-kappaB. Chromatin immunoprecipitation revealed that phosphorylated c-jun, but not NF-kappaBp65 bound to the natural IL-8 promoter. Production of endothelial IL-8 and simultaneous modulation of NF-kappaB in response to hydrolyzed LDL might serve to protect the vessel wall and promote silent removal of the insudated lipoprotein.

Pore-forming Staphylococcus aureus alpha-toxin triggers epidermal growth factor receptor-dependent proliferation.

Staphylococcal alpha-toxin is an archetypal killer protein that homo-oligomerizes in target cells to create small transmembrane pores. The membrane-perforating beta-barrel motif is a conserved attack element of cytolysins of Gram-positive and Gram-negative bacteria. Following the recognition that nucleated cells can survive membrane permeabilization, a profile of abundant transcripts was obtained in transiently perforated keratinocytes. Several immediate early genes were found to be upregulated, reminiscent of the cellular response to growth factors. Cell cycle analyses revealed doubling of S + G2/M phase cells 26 h post toxin treatment. Determination of cell counts uncovered that after an initial drop, numbers increased to exceed the controls after 2 days. A non-lytic alpha-toxin mutant remained without effect. The alpha-toxin pore is too small to allow egress of cytosolic growth factors, and evidence was instead obtained for growth signalling via the epidermal growth factor receptor (EGFR). Inhibition of the EGFR or of EGFR-proligand-processing blocked the mitogenic effect of alpha-toxin. Western blots with phospho-specific antibodies revealed activation of the EGFR, and of the adapter protein Shc. Immediate early response and proliferation upon transient plasma membrane pore formation by bacterial toxins may represent a novel facet of the complex interaction between pathogen and host.

Differential role of p38 mitogen activated protein kinase for cellular recovery from attack by pore-forming S. aureus alpha-toxin or streptolysin O.

Following the observation that cells are able to recover from membrane lesions incurred by Staphylococcus aureus alpha-toxin and streptolysin O (SLO), we investigated the role of p38 in this process. p38 phosphorylation occurred in response to attack by both toxins, commencing within minutes after toxin treatment and waning after several hours. While SLO reportedly activates p38 via ASK1 and ROS, we show that this pathway does not play a major role for p38 induction in alpha-toxin-treated cells. Strikingly divergent effects of p38 blockade were noted depending on the toxin employed. In the case of alpha-toxin, inhibition of p38 within the time frame of its activation led to disruption of the recovery process and to cell death. In contrast, blockade of p38 in SLO permeabilized cells did not affect the capacity of the cells to replenish their ATP stores.