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BIOCHEMICAL SOCIETY TRANSACTIONS
Gregoriadis, G., Leathwood, P. D. & Ryman, B. E. (1971) FEBSLett. 14,95-99
Kotoulas, 0.&Phillips, M. J. (1971) Amer. J. Puthol. 63,l-22
Phillips, M. J., Unakar, N. J., Doornewaard, G. & Steiner, J. W. (1967) J. Ultrustruct. Res.
18,142-165
The Inability of Macrophages to Digest Liposomes Containing
a High Proportion of Cholesterol
SHEENA M. JOHNSON
Division of Immunology, Clinical Research Centre, Warford Road,
Harrow, Middx. HA1 3UK, U.K.
Macrophages will ingest and break down many foreign bodies, but they do not damage
other normal cells with which they are surrounded. The purpose of this work is to find
out whether there are any specific properties of lipid bilayers which will prevent their
destruction by macrophages.
The liposomes were made of phosphatidylcholine, with or without cholesterol, and
contained trapped "NaCl. Two types of liposome were used, small unilamellar liposomes 25-5Onm diameter (depending on the amount of cholesterol) and larger multilamellar liposomes in aggregates about 10pm in diameter. The preparation of the small
liposomes, by sonication, is described in Johnson (1973). The multilamellar liposomes
were prepared by the vigorous mechanical shaking of 15pmol of phospholipid in 0.5 ml
of saline containing 15pCi of 22NaC1and buffered at pH7.6 (20°C) with 15m-TrisHCl. After 20min of violent agitation the liposomes were left at 37°C for 2h. To remove
untrapped label, the liposomes were passed over 3 g of hydrated Sephadex G-50,prepared
in nonradioactive buffered saline. They were then spun at 700g for IOmin, and the
supernatant was discarded. This removed any small unilamellar liposomes. The
liposomes were resuspended in a little Tris-saline, and samples were counted for
radioactivity and analysed for phosphate by McClare's method (1971). The liposomes
were diluted with Fisher's medium at pH6.7, containing penicillin and streptomycin.
were spun down, as before, resuspended and diluted to the required absorbance.
CBA mouse peritoneal macrophages were used in two different experimental systems.
The first method avoided liposome oxidation, whereas the second gave a better defined
cell population. In the first method, peritoneal exudate cells were washed three times with
Fisher's mediumcontainingantibiotics, then mixed with the liposomes. Portions (1 ml) of
themixture were put into a lOcm 8/32Visking dialysis tubing bag, which was surrounded
by 8ml of the medium. The tube was then incubated at 37"C, and the diffusate counted
for radioactivity at the end of the experiment. In the second method peritoneal exudate
cells were put into 3ml cell culture wells, without washing. After 0.5 h at 37°C they were
washed three times with Fisher's medium, containing antibiotics, and allowed to incubate for 2h at 37°C in COz+air (5:95) before the liposomes were added. At the end of
the experiment the liposomes were removed from the wells, put in Visking dialysis bags
and dialysed against 8ml of medium at room temperature for 45min. The diffusate was
counted for "Na. After checking cell survival, the wells were washed with 1% Triton
X-100 or 70% ethanol to release trapped "Na. Results were expressed as the percentage
of label released.
Experiments showed that the release of trapped label from the large liposomes was
strongly inhibited by 0.1 mwcyanide, but virtually unaffected by 10,ug of cytochalasin
B/ml and 1m-fluoride, a result consistent with a classical pinocytosis mechanism of
ingestion. The uptake of the small liposomes was less sensitive to cyanide.
Fig. 1 shows the time-course for the release of label from the large multilamellar
liposomes with and without cholesterol, with the use of the cell culture wells. It can be
seen that macrophages are unable to release "Na from the cholesterol-containing
liposomes. This result was obtained for liposomes containing a phospholipid/cholesterol
1975
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Fig. 1. Release of trapped zzNa from phosphatidylcholine liposomes, with and without a
1:1 molar ratio of cholesterol
Multilamellar phosphatidylcholineliposomes (0.1pmol) and 1:1 phosphatidylcholinecholesterol multilamellar liposomes (0.094pmol) were incubated with 2 x lo6 unstimulated CBA mouse peritoneal macrophages in Fisher's medium. A, Phosphatidylcholine
liposomes+macrophages; 0, phosphatidylcholineliposomes alone; A, phosphatidylcholins-cholesterol liposomes+maaophages; 0, phosphatidylcholin~holesterol
liposomes alone.
molar ratio above 1:0.43. Essentially similar results were obtained with the small
liposomes, and the presence of 10% foetal calf serum in themediummadeno qualitative
difference.
Preliminary results indicate that the inability of the macrophagesto release label from
liposomescontainingcholesterolmay be partially due to afailure to digest the liposomes.
At the end of the experiment only a negligible 0.1 % of the phosphatidylcholineliposoma were released by lysis of the macrophages whereas 1.2 % of the cholesterol liposome were found in the cells. CeII plasma membranes have a higher proportion of cholesterol than 1:0.43, so their lipid bilayer membranes should not be attacked in the
absence of more specific effects due to membrane protein.
Johnson, S.M.(1973)Biochim. Biophys. Acta 307,2741
McClare, C.W.F. (1971)Anal. Biochem. 39,527-530
The Identificationof Actin Associated with Pig Platelet
Membranes and Granules
DAVID G. TAYLOR, ROSEMARY J. MAPP and NEVILLE CRAWFORD
Department of Biochemistry, University of Birmingham, P.O. Box 363,
Edgbaston, Birmingham B15 2TT, U.K.
The presenceof contractile proteins in cells other than muscle is well established and the
involvement of these proteins in cell motile activities is generally accepted (for review,
see Pollard & Weihing, 1974). Bettex-Galland & Luscher (1959) isolated an actomyosinlike complex from human blood platelets and, subsequently, Grette (1962) extracted a
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