Effect of agents that produce membrane disorder on lysis of erythrocytes by complement.

Abstract

To evaluate the effect of membrane lipid acylchain packing on the efficiency of cell lysis by complement, we have studied membrane modulation by 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) and by myristoleyl alcohol, the cis isomer of a C14:1 aliphatic alcohol. These substances are known to increase the membrane lipid disorder by virtue of the bend in their acyl chains, which is believed to loosen the phospholipid acyl-chain packing. We have found that both of these compounds markedly enhance the lysis of erythrocytes by the terminal complement proteins C5b-9. The enhancing effect by A2C is operative in the formation oferythrocytes carrying complement components C5b, C6, and C7, as well as in the subsequent reactions with complement components C8 and C9. We have also found that A2C-treated erythrocytes bind C5b6 to a measurable extent, whereas untreated erythrocytes do not. We attribute this to a shift in the partition equilibrium of C5b6 toward membrane association, which would improve lytic efficiency. The increase of membrane lipid disorder by these agents would also be expected to increase insertion of hydrophobic peptides from C7, C8, and C9, with consequent gain in lytic efficiency. Treatment of erythrocytes with sublytic doses of NaDodSO4 or Triton X-100 did not enhance lysis by C5b-9 appreciably, suggesting that enhancement of lysis by C5b-9 is not a general property of amphiphiles. Membrane attack by complement (C) is initiated when the com-.ponent C5 is cleaved into C5a and C5b by the C5 convertases of either the classical or alternative activation pathways. The subsequent interactions among the terminal complement proteins (C5b-C9) are accompanied by exposure of hydrophobic peptides (1-3). If this occurs in the immediate vicinity of a bilayer membrane, some ofthe exposed peptides became inserted in the lipid bilayer (4-6) and are assembled into channels that permit ion flow across the membrane (7-11). In the case ofcells that are susceptible to colloid-osmotic disruption, this leads to cytolysis. The processes of insertion and channel formation are affected by the chemical properties of the membrane (12)(13)(14)(15)(16) Chemicals. A2C (Makor, Jerusalem, Israel) was dispersed in 5 mM sodium barbital/142 mM sodium chloride, pH 7.4 (NaCl/ B). This dispersion was sonicated in a bath sonicator at room temperature until a homogeneous turbid colloidal suspension was obtained ('-3-5 min). Initially, 2 ,ul of A2C was dispersed in 5 ml of the NaCl/B to make a 0.1% stock suspension.Myristoleyl alcohol (Sigma) or its trans isomer (Nu Check Prep, Elysian, MN) (an unsaturated bond at C7-C8 position) were dissolved in chloroform to a concentration of 1%. This solution was mixed with NaCl/B and sonicated with a 1-cm diameter probe at a 40% power setting (Biosonick IV) for 3-5 min at 0°C.Triton X-100 (Sigma) or NaDodSO4 (BDH, Poole, England) were dissolved initially in NaCl/B to final concentrations of0.1 and 0.05 mM, and further dilutions were later made to determine the sublytic c...

Influence of acyl chain fluidity on the lipopolysaccharide-induced activation of complement

“…Membrane modulation by 2-(2methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)octanoate and by myristoleyl alcohol, which are known to increase membrane disorder by loosening the phospholipid acyl chain packing, was found to increase C lysis of erythrocytes by the terminal complement proteins C5b-9. 11 From a mechanistic view, assembly of the membrane attack complex involves the incorporation of hydrophilic molecules into a macromolecular amphiphilic complex capable of integrating into lipid bilayers. This integration should be much simpler when membrane fluidity is sufficiently high.…”

“…8,10 The influence of membrane disorder on C activation has also been addressed by Shin et al who found in a reactive lysis assay using sheep erythrocytes that 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate and myristoleyl alcohol, which are known to increase membrane disorder, enhance C lysis. 11 Performing electrical measurements on a reconstitution model of the outer membrane of Gram-negative bacteria as asymmetric planar LPS/phospholipid bilayer, we were able to characterize pore formation by C in a very direct manner. 12,13 Thus, we recently characterized the structural requirements of the sugar moiety of the glycolipids for C activation.…”

Bactericidal and bacteriolytic activity of serum against gram-negative bacteria.

“…The degree of disruption of phospholipidcholesterol liposomes varies inversely with the concentration of membrane cholesterol (275), and there is a correlation between susceptibility of sheep erythrocytes to complement attack and the ratio of membrane lecithin to sphingomyelin (171), suggesting that increased membrane fluidity enables a more efficient assembly and integration of MACs on phospholipid membranes. Incorporation of the fluidizing and fusogenic agent 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate into sheep erythrocyte membranes loosens phospholipid acylchain packing and thus increases membrane lipid disorder; cells treated in this way become extremely susceptible to complement lysis (274).…”

Optimized Solid‐Phase‐Assisted Synthesis of Oleic Acid Containing siRNA Nanocarriers

“…[6] Cationic lipids containing cis-unsaturated FAs can promote membrane lipid disorders by interacting with anionic phospholipids. [7] The fusion between two lipid membranesi sm ediated by al amellar-to-hexagonal inverted phase transition. [8] This process is promoted by cone-shapedc ationic lipids, where the cross-sectional area of the hydrophilic head groups is smaller than that of the hydrophobic tails.…”

“…Well‐known cationic lipids like DOPE, DOTMA and DOSMA contain the cis ‐unsaturated fatty acid (FA) oleic acid and were first used to efficiently delivery DNA and mRNA in 1987 and 1989, respectively . Cationic lipids containing cis ‐unsaturated FAs can promote membrane lipid disorders by interacting with anionic phospholipids . The fusion between two lipid membranes is mediated by a lamellar‐to‐hexagonal inverted phase transition .…”