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Naturally occurring antibodies to

cholesterol: a new theory of LDL
cholesterol metabolism
Carl R. Alving and Nabila M. Wassef
Although low-density lipoprotein
(LDL) receptors regulate the
disposition of two-thirds of
hirteen Nobel prizes awarded
Receptor-independent pathways of
LDL degradation
for research on the structure,
circulating serum LDL cholesterol,
The LDL receptor is characterized by highconformation, synthesis and renon-LDL receptor mechanisms
affinity binding of LDL, with a marked prefceptor cell biology of cholesaccount for removal of one-third.
erence for binding LDL rather than HDL
terol and related molecules has prompted
(Ref. 11). However, LDL receptor-indepenthe comment that cholesterol is the most
Here, Carl Alving and Nabila
dent uptake of radiolabeled LDL can also be
highly decorated small molecule in biolWassef propose that naturally
1. The discovery of the low-density
readily demonstrated in vivo in two types of
ogy
occurring antibodies to cholesterol
lipoprotein (LDL) receptor-mediated pathsystems: (1) genetically deficient patients or
way for cholesterol homeostasis by Brown
rabbits in which LDL receptors are absent;
in normal human plasma also
and Goldstein has led to an armamentarium
and (2) normal humans or animals in which
contribute to LDL cholesterol
of successful drugs and drug strategies
the radiolabeled LDL that is injected has
aimed at lowering LDL, very low-density
been chemically modified to block binding
turnover by opsonizing LDL and
lipoprotein (VLDL) and intermediate-dento the LDL receptor1,12. The above models
other lipoproteins containing bad
suggest that the LDL receptor is normally resity lipoprotein (IDL) cholesterol levels by
cholesterol for removal by
sponsible for the removal of only approxiinhibiting cholesterol biosynthesis and up14
mately two-thirds of circulating LDL cholesregulating LDL receptor expression . An
complement receptors.
ambitious goal has now been set for the
terol1,12. However, it should be noted that
elimination of coronary disease early in the
rabbits and other nonprimates lack CR1, and
next century5. Despite the panoply of cholesterol-lowering drugs, the important process of uptake of immune complexes by immune
the achievement of this goal is a challenge that could still benefit adherence to erythrocytes and shuttling to scavenger cells does not
from further theoretical and therapeutic insights6,7.
occur9. Therefore, nonprimates might not be suitable as models for
Could immunological mechanisms play a role in cholesterol determining the actual cellular mechanisms responsible for receptormetabolism? In this article we propose that a specific immunological independent uptake of LDL by macrophages in humans.
mechanism, opsonization of LDL, VLDL and IDL cholesterol by the
binding of naturally occurring antibodies to cholesterol, results in activation of complement (C) and binding of C split products (mainly Antibodies to cholesterol
C3b) to the lipoprotein. Uptake of the circulating lipoproteinanti- Experimentally induced
cholesterolC3b immune complexes by cells expressing C3b receptors Cholesterol is an extremely immunogenic molecule when combined
then affects the normal turnover and distribution of LDL cholesterol with the proper adjuvant and carrier13. Protein-free liposomes
in the plasma (Fig. 1). It is likely that most of the initial uptake occurs loaded with highly purified non-oxidized cholesterol, and also conthrough binding to complement receptor type 1 (CR1) molecules on taining a potent adjuvant (lipid A) derived from Gram-negative bacerythrocytes8,9, which bind immune complexes containing C3b terial endotoxin, serve as an efficient immunizing formulation for
through the phenomenon known as immune adherence, after induction of polyclonal and monoclonal antibodies, as detected by
which the immune complexes are shuttled by erythrocytes to ELISA with pure cholesterol1315. The purity of the cholesterol antimacrophages9. Erythrocytic CR1 is a major site, both for removal of gen is assured by recrystallization three times from hot ethanol.
circulating immune complexes in primates and for downregulation The product then has the melting point of cholesterol and is free of
of further C activation9. The current theory predicts that im- detectable oxidation products (,0.04% peroxides) on thin layer
munomodulation of cholesterol is specific to LDL, VLDL and IDL chromatography, as determined by Wurster dye using 25-hydroxy(lipoproteins containing bad cholesterol) rather than to high- cholesterol as a standard14. As one indication of the effectiveness of
density lipoprotein (HDL) cholesterol (good cholesterol) because the purification process, it was found that after each recrystallization
the anticholesterol antibodies do not bind HDL cholesterol10. Exper- the cholesterol showed progressive improvement when used as an
imental evidence supporting a natural role for immunomodulation antigen in microtiter wells for detecting cholesterol antibodies14. Antibodies to cholesterol and, to a lesser extent, phospholipids, are also
of LDL cholesterol is provided below.

0167-5699/99/$ see front matter 1999 Elsevier Science Ltd. All rights reserved.

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apoB
induced in most mice that are injected intraperitoneally with an oil such as silicone16.
Presumably, the silicone recruits phospholipids and cholesterol from cells or cellular
debris to form an emulsion, which serves as
a carrier for induction of antibodies to cholesterol and phospholipids.

Cholesterol

Binding of naturally-occurring
cholesterol antibodies to LDL

LDL in
plasma
LDL
Hepatocyte

Antibody

C3

CR1

RBC

Naturally occurring

In view of the impressive immunogenicity of

LDL receptor
cholesterol promoted by lipid A endotoxin, a
CR1
C3
molecule that is found ubiquitously in the
b
C3b
environment, it is perhaps not surprising
that naturally occurring antibodies to cholesterol, both IgM and IgG, are found in humans and in many animals13,17,18. In one
study, we determined that each of 742 normal human volunteers had detectable antiCR1
bodies to cholesterol, with levels ranging
from very high to very low (Fig. 2)19. Similarly, Avila et al. found naturally occurring
Kupffer
antibodies to cholesterol in sera from each
cell
of 100 normal human control subjects and
also from 127 patients with chronic Chagas
Fig. 1. Immunomodulation of LDL. A proposed immunological mechanism is shown whereby antidisease20.
bodies to cholesterol bind to LDL (or VLDL or IDL), causing opsonization by complement and reExperimental induction of murine antimoval of the lipoprotein from the blood by macrophages. As discussed in the text, the initial binding
bodies to cholesterol can be achieved by
of the LDLantibodycomplement complex probably occurs on erythrocytes through the process of imusing liposomes containing high concenmune adherence to CR1, with subsequent shuttling of the immune complex to the macrophages. This
trations of cholesterol and lipid A. This
results in LDL receptor-independent uptake of lipoprotein. Abbreviations: apo B, apolipoprotein B;
model would then predict that naturally
C, complement; CR1, complement receptor type 1; IDL, intermediate-density lipoprotein; LDL, lowoccurring antibodies to cholesterol could be
density lipoprotein; VLDL, very low-density lipoprotein.
induced in humans by the large amount of
endotoxin that exists everywhere in the environment. Because Gram-negative bacteria themselves do not con- lack specificity for HDL (Ref. 10). The antibodies bind to cholesterol
tain cholesterol, a rich environmental supply of cholesterol would extracted from HDL, but not to intact HDL (Ref. 10). The binding of
also be required for antibody induction. Although the source of chol- the anticholesterol antibodies to LDL, VLDL and IDL, also observed
esterol antigen for inducing naturally occurring antibodies is un- in rabbits22, can be explained by the relatively low protein (apoproknown, it could come from localized sites of infection and inflam- tein) to cholesterol ratios found at the surface of these lipoproteins
mation, where cellular debris has accumulated, or from infection or (protein : cholesterol based on % of lipoprotein mass 5 1.4 for VLDL,
colonization of bacteria in the skin, where a very rich supply of free 1.9 for IDL and 2.1 for LDL)23. By contrast, a relatively high protein
cholesterol is present. In accordance with the concept of a microbial to cholesterol ratio is found at the surface of HDL (3.5 for HDL-E, 8.4
mechanism, antibodies to cholesterol (IgG) have been induced in for HDL2 and 18.7 for HDL3)23, and appears to have an inhibitory
rabbits by injection of cultured Mycoplasma cells, several species of effect, perhaps by simple steric hindrance, charge effects or hydrowhich are common inhabitants of the human oral cavity and lower phobic blocking, on the binding of the antibody molecule to the
extremely small 3b-hydroxyl group of free cholesterol at the surface
urogenital tract21.
of the HDL particle. Naturally occurring antibodies to cholesterol
that differentially bind to LDL may also explain the previous finding
Specificity of cholesterol antibodies
that immunoglobulins (IgM and IgA) are the major LDL-binding
Murine antibodies to cholesterol specifically recognize the 3b- proteins in normal human plasma24.
hydroxy group of cholesterol and because of this they also crossreact
When compared with the much larger polar headgroups of
with certain cholesterol analogs (including oxidized analogs) that phospholipids and glycolipids, and even larger interfering struccontain the 3b-hydroxy group10. Interestingly, murine anticholes- tures of membrane-associated proteins, the polar hydroxyl region of
terol antibodies bind specifically to human LDL, VLDL and IDL, but cholesterol is both dwarfed in size and located at a protected site at

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ratios25. This steric interference on binding of soluble proteins to

cholesterol explains why antibodies to cholesterol do not have a
detrimental effect on cell membranes, and implies that antibodies
to cholesterol might have easy access to the higher concentrations of
relatively exposed cholesterol on the surface of LDL, VLDL and
IDL.

Immunomodulation of LDL cholesterol by antibodies

to cholesterol
Based on the properties summarized in Box 1, our laboratory has
been intrigued by the question of whether antibodies to cholesterol
could have the ability to influence serum levels of LDL, VLDL and
IDL. To address this issue, we immunized rabbits with cholesterolladen liposomes containing lipid A to induce (or enhance the natuFig. 2. Anticholesterol antibodies in normal human sera. Dilutions (1:50) ral titers of) antibodies to cholesterol, and we then fed a cholesterolof each of 742 human sera were tested in triplicate by ELISA with purified rich diet to increase the levels of LDL, VLDL and IDL (Ref. 22).
non-oxidized cholesterol as antigen and mixed-affinity purified antihuman Because of the extreme sensitivity of rabbits to dietary cholesterol,
IgG, IgM and IgA (H and L chains). Titration of a standard serum used for this experimental protocol causes massive increases of cholesterol
21
21
comparison was performed with each group of ~15 volunteer sera (inset from normal levels of ,75 mg dl to as much as 2500 mg dl or
shows mean 6 SD of 52 assays of the standard serum). Binding was not more within weeks (Fig. 4). For comparison, the normal total choles21
observed when cholesterol was omitted. Figure reproduced, with permis- terol level in human serum is ~100200 mg dl . In this model, animals immunized with cholesterollipid A liposomes exhibited insion, from Ref. 19.
creased levels of anticholesterol antibodies and showed a marked
the lowest point at the surface of the lipid bilayer of the cell mem- delay in the rise of serum cholesterol, with values as much as 36%
brane (Fig. 3)16. Steric hindrance to interaction with cholesterol also lower than those observed in the control rabbits. Perhaps most draapplies to oxidizing enzymes, as shown by the observation that ac- matic was the absolute difference of 979 mg dl21 of cholesterol that
cessibility to liposomal cholesterol or viral cholesterol by cholesterol occurred as a result of the delayed rise in cholesterol levels in the imoxidase is greatly facilitated by increased cholesterol : phospholipid munized animals compared with non-immunized animals (at 10
weeks in Fig. 4). This large absolute differential between the two groups suggests that
the mechanism of immunomodulation has a
great capacity for LDL cholesterol reduction.
Importantly, the degree of aortic atherosclerotic plaque formation was also significantly reduced in the above immunized rabbits, indicating that immunomodulation of
cholesterol levels resulted in physiological
benefits. Independent studies in which rabbits were immunized with oxidized LDL
and then made hypercholesterolemic also reported diminished atherosclerosis when
compared with non-immunized rabbits26,27.
Although effects on LDL cholesterol levels
were not examined in the latter studies, as
mentioned earlier, certain oxidized cholesterol analogs that retain the 3b-hydroxy
group have the capacity to bind to anticholesterol antibodies. By the same reasoning,
immunization with oxidized cholesterol
analogs that contain the 3b-hydroxy group
Fig. 3. Comparative sizes of cholesterol, phosphatidylcholine (PC), ganglioside GM1, glycophorin and might also be expected to induce antibodies
a peripheral globular protein in a lipid bilayer membrane. Figure reproduced, with permission, from that would crossreact with cholesterol and
influence LDL cholesterol levels.
Ref. 16.

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Box 1. Some initial concepts suggesting that naturally occurring

antibodies to cholesterol might contribute to normal uptake of LDL
In the above study, the ability of the anti The LDL receptor accounts for only approximately two-thirds of the LDL normally
bodies to retard the elevation of levels of
removed from plasma in humans, with the rest removed by scavenger cells
LDL, VLDL and IDL eventually became
through a non-characterized mechanism
saturated and was overcome (at 12 weeks in

Immunoglobulins (IgM and IgA) are the major LDL-binding proteins in normal
Fig. 4) by the increasing burden of serum
human
plasma
cholesterol caused by continued feeding of

Naturally
occurring antibodies to cholesterol, both IgG and IgM, are present
22
the high cholesterol diet . Nonetheless, the
ubiquitously
in normal human serum
experiments with the rabbit model showed
Polyclonal and monoclonal antibodies to cholesterol bind to LDL, VLDL and IDL,
that antibodies to cholesterol have the capacbut not to HDL
ity to bind and remove large amounts of

The origins of naturally occurring antibodies to cholesterol may relate to

LDL cholesterol from the circulation, thus
coexposure
to Gram-negative bacteria, bacterial endotoxins, or certain other
suggesting that this process could occur
microorganisms
because cholesterol is very immunogenic in such conditions
under normal conditions.

Antibodies
to
cholesterol,
whether induced or naturally occurring, do not have any
If this mechanism does play a role, one
apparent
deleterious
effect
on normal cells or tissues
would expect to observe circulating immune
complexes containing LDL, VLDL and IDL
Abbreviations: IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density
in association with anticholesterol antibodlipoprotein; VLDL; very low-density lipoprotein.
ies in the plasma under normal conditions.
In support of this, immunoglobulins (mainly
IgM and IgA) have been reported to be the major LDL-binding pro- the observation that immunosuppression is associated with hyperteins in normal human plasma24. By the same reasoning, antibodies cholesterolemia.
bound to LDL cholesterol and free (unbound) antibodies should
This theory raises the likelihood that LDL cholesterol levels are
exist in equilibrium in the plasma. Indeed, the ability to detect anti- controlled in part by well-known immunological properties of antibodies to cholesterol, either by ELISA (Refs 15, 17, 19, 20, 22) or bodies, including titer, isotype, affinity and specificity, along with
by complement-dependent immune damage to liposomes laden
with cholesterol (another method for detecting anticholesterol antibodies)22, is indirect evidence that such an equilibrium does exist.

Effects of immunosuppression on serum cholesterol

levels
If the above theory of immunomodulation of serum LDL levels by
naturally occurring antibodies to cholesterol is correct, then it follows that immunosuppression would be associated with decreased
removal of LDL cholesterol by antibodies and elevation of serum
cholesterol in immunosuppressed individuals. This is indeed observed. The administration of cyclosporine and steroids, drugs that
are utilized as a regimen of immunosuppression after organ transplantation, is often associated with significant hypercholesterolemia,
a condition that can result in generalized cardiovascular disease and
accelerated graft atherosclerosis2830. Although as many as 71% of
transplanted individuals are afflicted with hypercholesterolemia29,
many of whom require resultant treatment, the cause of the raised
cholesterol is still unknown.

Implications of the theory

The multiple lines of evidence that support the concept of immunomodulation of serum LDL cholesterol levels include: the
ubiquitous existence of antibodies to cholesterol in human serum;
differential binding of the antibodies to LDL, VLDL and IDL, but not
to HDL; the blocking of diet-induced hypercholesterolemia in rabbits immunized with cholesterol; the demonstration of immunoglobulin as a major LDL-binding protein in human serum; and

Fig. 4. Time course of diet-induced hypercholesterolemia in rabbits immunized to induce raised levels of antibodies to cholesterol. The rabbits were
immunized at 0, 2, 4 and 6 weeks with liposomes containing both lipid A as
adjuvant and a high concentration of cholesterol (71%) as antigen. Starting
at week 5, the animals were fed a diet containing 1% cholesterol. In this
model, nearly all of the hypercholesterolemia is due to LDL, VLDL and IDL.
Each bar represents the mean 6 SEM (n 5 4 for immunized and control
groups). *p 5 0.004; **p ,0.05; ***p 5 0.001. Figure reproduced, with
permission, from Ref. 22. Abbreviations: IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein.

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the ability of immune complexes to bind to receptors, such as the

erythrocytic CR1 receptor. It is also likely that LDL cholesterol normally exists as immune complexes with anticholesterol antibodies in
the plasma, and the apparent titers of free antibodies could therefore
be influenced by the activities of the LDL receptor, or vice versa.
Finally, if regulation of LDL cholesterol is at least partly controlled
by antibodies to cholesterol, this raises the possibility that increasing
the titer of the antibodies by direct immunization with cholesterol
could have a beneficial effect as a unique new strategy for lowering
serum LDL cholesterol levels31.

13 Alving, C.R. and Swartz, G.M., Jr (1991) Crit. Rev. Immunol. 10, 441453
14 Swartz, G.M., Jr, Gentry, M.K., Amende, L.M., Blanchette-Mackie, E.J.
and Alving, C.R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 19021906
15 Aniagolu, J., Swartz, G.M., Dijkstra, J., Madsen, J.W., Raney, J.J. and
Green, S.J. (1995) J. Immunol. Methods 182, 8592
16 Alving, C.R., Wassef, N.M. and Potter, M. (1996) Curr. Top Microbiol.
Immunol. 210, 181189
17 Alving, C.R., Swartz, G.M., Jr and Wassef, N.M. (1989) Biochem. Soc.
Trans. 17, 637639
18 Wassef, N.M., Johnson, S.H., Graeber, G.M. et al. (1989) J. Immunol. 143,
29902995
19 Alving, C.R., Swartz, G.M., Jr, Wassef, N.M. et al. (1995) in Atherosclerosis

We thank T. Scharton-Kirsten and J. Szebeni for extremely valuable discussions and reviews of the manuscript.

X (Woodford, P., Davignon, J. and Sniderman, A., eds), pp. 944948,

Elsevier Science
20 Avila, J.L., Rojas, M. and Avila, A. (1996) Clin. Exp. Immunol. 103, 4046

Carl Alving (carl.alving@na.amedd.army.mil) and Nabila Wassef are at

the Walter Reed Army Institute of Research, Dept of Membrane Biochemistry,
Washington, DC 20307-5100, USA.

21 Watanabe, T. and Kumazawa, T. (1991) Infect. Immun. 59, 22002202

22 Alving, C.R., Swartz, G.M., Wassef, N.M. et al. (1996) J. Lab. Clin. Med.
127, 4049
23 Kostner, G.M. and Laggner, P. (1989) in Human Plasma Lipoproteins

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