Biophysical characterization of the interaction of high-density
lipoprotein (HDL) with endotoxins
Klaus Brandenburg
1
, Gudrun Ju¨ rgens
1
,Jo¨ rg Andra¨
1
, Buko Lindner
1
, Michel H. J. Koch
2
, Alfred Blume
3
and Patrick Garidel
3
1
Forschungszentrum Borstel, Biophysik, Borstel, Germany;
2
European Molecular Biology Laboratory, Hamburg Outstation, EMBL
c/o DESY, Hamburg, Germany;
3
Martin-Luther-Universita
¨
t Halle/Wittenberg, Institut fu
¨
r Physikalische Chemie, Halle, Germany
The interaction of bacterial endotoxins [lipopolysaccharide
(LPS) and the Ôendotoxic principleÕ lipid A], with high-den-
sity lipoprotein (HDL) from serum was investigated with a
variety of physical techniques and biological assays. HDL

addition of HDL. These data allow to develop a model of
the [endotoxin]/[HDL] interaction.
Keywords: endotoxin conformation; high density lipopro-
teins (HDL); lipopolysaccharides; Fourier-transform infra-
red spectroscopy.
Bacterial lipopolysaccharides (LPS) belong to the most
potent stimulators of the immune system and play an
important role in the pathogenesis and manifestation of
Gram-negative infections, in general, and of septic shock,
in particular, and are thus called endotoxins. The
mechanism of endotoxin interaction with different target
cell structures are still largely unknown and only limited
data are available on the detailed mode of binding of
endotoxins to various endogenous proteins, which are
important with regard to combat invading microorgan-
isms and to transport and neutralize free endotoxin.
Among the humoral factors which are important LPS-
binding molecules are serum lipoproteins. It was sugges-
ted that sequestering of LPS by lipid particles may form
an integral part of humoral detoxification [1]. Lipo-
proteins are water-soluble complexes with a neutral core,
surrounded by a phospholipid layer that contains
cholesterol and one or more ÔapolipoproteinsÕ.Theyserve
as ligands for cell membrane receptors, as cofactors for
enzymes, and can dock lipopolysaccharide-binding pro-
teins. They are classified as very-low density, low-density
and high-density lipoproteins (HDL) according to their
buoyant density. The primary function of these lipo-
proteins is to transport lipids, cholesterol and cholesteryl
esters in blood and the lymphatic system. HDL moreover

were observed for the latter, using the attenuated total
reflectance (ATR) method. To obtain information about
the phase transition enthalpy changes of the endotoxins,
differential scanning calorimetry in the absence and
presence of HDL was carried out. Also, with FTIR the
influence of endotoxin binding on the secondary structure
of the protein part of HDL, apolipoprotein A-I (apoA-I)
was observed. The effect of HDL on the surface charge of
the endotoxin aggregates was studied by applying zeta
potential measurements, which also enabled an estimate
for the binding saturation to be made. The aggregate
structure and, with that, the conformation of the lipid A
part of LPS was studied by small-angle X-ray diffraction.
With fluorescence resonance energy transfer experiments,
information about the influence of HDL on the inter-
calation of LPS and LBP, and the intercalation of the
lipoprotein itself into phospholipid target membranes
could be given. Finally, in biological experiments the
ability of the endotoxin and [endotoxin]/[HDL] complexes
to induce cytokine production in mononuclear cells and to
activate the Limulus amebocyte lysate (LAL) clotting
cascade was measured. Thus, it was possible to charac-
terize the binding of HDL to the endotoxins profoundly
and to get insight into the mechanisms of the reduction of
the LPS-induced cytokine production in human mono-
nuclear cells.
MATERIALS AND METHODS
Lipids and reagents
Lipopolysaccharide from the deep rough mutant Re
Salmonella minnesota (R595) was extracted by the phenol/

toxins, HDL was prepared in buffer made either from H
2
O
or D
2
O incubated at 37 °C for 30 min, and lipid dispersions
prepared as described above were added in appropriate
amounts, and further incubated at 37 °C for 15 min.
Afterwards, 10 lL of these dispersions were spread on a
CaF
2
infrared window, and the excess water was evaporated
slowly at 37 °C.
FTIR spectroscopy
The infrared spectroscopic measurements were performed
on a 5-DX FTIR spectrometer (Nicolet Instruments,
Madison, WI, USA) and on an IFS-55 spectrometer
(Bruker, Karlsruhe, Germany). The lipid samples were
placed in a CaF
2
cuvette with a 12.5-lm Teflon spacer.
Temperature-scans were performed automatically between
10 and 70 °C with a heating-rate of 0.6 °CÆmin
)1
.Every
3 °C, 50 interferograms were accumulated, apodized, Fou-
rier transformed and converted to absorbance spectra. For
strong absorption bands, the band parameters (peak
position, band width, and intensity) were evaluated from
the original spectra, if necessary after subtraction of the

)1
. The measurements were performed at 26 °C, the
intrinsic instrument temperature, in some cases also at
37 °C.
Differential scanning calorimetry
LPS was dispersed in buffer at a concentration of
1mgÆmL
)1
. A liposomal lipid dispersion was obtained by
sonication for 10 min at 40 °C. After cooling to room
temperature, a defined amount of HDL was added to 1 mL
lipid dispersion and the sample was gently vortexed until
Ó FEBS 2002 HDL interaction with endotoxins (Eur. J. Biochem. 269) 5973
HDL was completely dissolved [9]. Differential scanning
calorimetry measurements were performed with a MicroCal
VP scanning calorimeter (MicroCal, Inc., Northampton,
MA, USA). The heating and cooling rate was 1 °CÆmin
)1
.
Heating and cooling curves were measured in the tempera-
ture interval from 10 to 100 °C. Three consecutive heating
and cooling scans were measured [10].
X-ray diffraction
X-ray diffraction measurements were performed at the
European Molecular Biology Laboratory (EMBL) outsta-
tion at the Hamburg synchrotron radiation facility HASY-
LAB using the double-focusing monochromator-mirror
camera X33 [11]. Diffraction patterns in the range of the
scattering vector 0.07 < s <1nm
)1

2
)/a]
1/2
, where hkl are Miller indices of the
corresponding set of plane.
Zeta potential
Zeta potentials were determined with a Zeta-Sizer 4
(Malvern Instr., Herrsching, Germany) at a scattering angle
of 90° from the electrophoretic mobility by laser-Doppler
anemometry as described earlier [15]. The zeta potential was
calculated according to the Helmholtz-Smoluchovski equa-
tion from the mobility of the aggregates in a driving electric
field of 19.2 VÆcm
)1
. It was determined for the endotoxins
(0.5 m
M
) at different HDL concentrations.
Isothermal titration calorimetry
Microcalorimetric experiments of HDL-binding to endo-
toxins were performed on an MCS isothermal titration
calorimeter (Microcal Inc., Northampton, MA, USA). The
endotoxin samples at a concentration of 0.25 mgÆmL
)1
,
prepared as described above, were filled into the microca-
lorimetric cell (volume 1.3 mL), and HDL at concentrations
up to 12 mgÆmL
)1
were loaded into the syringe compart-

[endotoxin]/[HDL] mixtures, human mononuclear cells
were stimulated with the latter and the IL-6 production of
the cells was determined in the supernatant.
Mononuclear cells were isolated from heparinized (20
IEÆmL
)1
) blood taken from healthy donors and processed
directly by mixing with an equal volume of Hank’s balanced
solution and centrifugation on a Ficoll density gradient for
40 min (21 °C, 500 g). The layer of mononuclear cells was
collected and washed twice in Hank’s medium and once in
serum-free RPMI 1640 containing 2 m
ML
-glutamine,
100 UÆmL
)1
penicillin, and 100 lgÆmL
)1
streptomycin. The
cells were resuspended in serum-free medium and their
number was equilibrated at 5 · 10
6
cellsÆmL
)1
. For stimu-
lation, 200 lLÆwell
)1
mononuclear cells (5 · 10
6
cellsÆmL

sulfuric
acid. In the color reaction, the substrate is cleaved
enzymatically, and the product was measured photometri-
cally on an
ELISA
reader (Rainbow, Tecan, Crailsham,
5974 K. Brandenburg et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Germany) at a wavelength of 450 nm and the values were
related to the standard. IL-6 was determined in duplicate at
two different dilutions and the values were averaged.
Determination of endotoxin activity by the
chromogenic
Limulus
test
Endotoxin activity of [LPS]–[HDL] mixtures at concentra-
tions between 10 lgÆmL
)1
and 10 pgÆmL
)1
was determined
by a quantitative kinetic assay based on the reactivity of
Gram-negative endotoxin with LAL [19], using test kits
from LAL Coamatic Chromo-LAL K (Chromogenix,
Haemochrom). The standard endotoxin used in this test
was from E. coli (O55:B5), and 10 EUÆmL
)1
corresponds to
1ngÆmL
)1
. In this assay, saturation occurs at 125 endotoxin

)
hydr.
1230–1220 cm
)1
and m
as
(diglucosamine)
1180–1150 cm
)1
, can be seen; the addition of HDL leads to
an intensity decrease in the band contours proportional to
the HDL concentration. From Fig. 1 it can be taken that
especially the intensity of the band component around
1190 cm
)1
increases as compared with that at lower
wavenumbers and becomes sharper. Additionally, the
component at 1170 cm
)1
for pure LPS is shifted to
approximately 1177 cm
)1
in the presence of HDL. These
results indicate that (i) besides the phosphate groups, the
sugar diglucosamine part in lipid A are also binding-sites for
HDL, and (ii) these vibrational bands are immobilized due
to HDL binding.
Gel to liquid crystalline (b«a) phase behavior
The b«a gel to liquid crystalline acyl chain melting behavior
was investigated with FTIR by evaluating the peak position

c
¼ 45 °C of pure lipid A is shifted
to 50 °C (data not shown). This observation reflects the
different number of negative charges and monosaccharide
units (LPS Re has four negative charges and four sugar
units, lipid A two of each) which may be connected with
different conformations of the molecules.
Differential scanning calorimetry measurements of the
interaction of LPS with HDL (Fig. 3) shows for pure LPS a
phase transition in accordance to that observed in Fig. 2.
Fig. 1. Infrared-ATR spectra in the range of the antisymmetric
stretching vibration of the negatively charged phosphate groups
m
as
(PO
2
–
)1210–1260 cm
)1
) and the diglucosamine ring vibration (see
arrows) of LPS at different [LPS]/[HDL] weight ratios. The spectra
were normalized by taking the band intensity of the symmetric
stretching vibration of the methylene groups m
s
(CH
2
)asstandard.
Fig. 2. Peak position of the symmetric stretching vibration of the
methylene groups m
s

the first and succeeding cooling scan. The thermograms of
the succeeding heating scan are slightly broader compared
with the 1st heating scan (Fig. 3A).
HDL was added to LPS at different concentrations
{[LPS]/[HDL] 1 : 0.25, 1 : 0.45, 1 : 0.6 and 1 : 1 (w/w)}. In
Fig. 3(B) representative thermograms for the sample at a
LPS/HDL 1 : 1 (w/w) ratio are plotted. The phase trans-
ition temperature of LPS is shifted from 31 °Cto 33 °C,
the half-width of the phase transition is increased
(T
1/2
¼ 7 °C) and the phase transition enthalpy is decreased
by  22%. The presence of HDL induces a broadening of
the coexistence range of the phase transition, especially for
the offset temperature which is shifted above 42 °C. The
phase transition as derived from the IR spectra from the
temperature dependence of m
s
(CH
2
) of the [LPS]/[HDL]
1 : 0.5 system revealed similar data: T
c
¼ 34 °Cand
T
1/2
¼ 8.5 °C. The heat-capacity curve of LPS/HDL ratio
develops a shoulder starting at  20 °C in the gel phase
indicating that HDL interacts with the gel phase LPS. This
is observed for all four investigated LPS/HDL concentra-

of [LPS]/[HDL] 1 : 1.5 (Fig. 4). Addition of HDL to the
preincubated [LPS]–[PMB] complex leads to almost the
same result as without HDL, and addition of PMB to
preincubated [LPS]–[HDL] causes a slightly attenuated
fluidizing effect as compared with LPS with PMB alone.
PMB, which binds much stronger to the LPS phosphates
than HDL, may displace HDL molecules from their binding
site, the lipid A phosphates.
These results are complemented by the data of the
dephospho-LPS Re and HDL systems (Fig. 5). Dephos-
pho-LPS Re has a T
c
of 45 °C,andinthecaseof
phosphates as the primary binding site no change of the
phase behavior of dephospho-LPS Re would be expected.
However, addition of HDL causes a fluidization parti-
cularly in the gel phase and in the transition range at a
Fig. 3. Differential scanning calorimetry heat
capacity curves of pure LPS Re (A), a mixture
of [LPS]/[HDL] at 1.1 : 1 w/w (B), and for pure
HDL (C). Heating and cooling curves were
measured in the temperature interval 10–
100 °C. Three consecutive heating and cooling
scans are presented (A,B) (h.s. heating-scan,
c.s. cooling scan) and first heating scan (C).
Fig. 4. Peak position of the symmetric stretching vibration of the
methylene groups m
s
(CH
2

d
|
¼ 5.13 nm and 2.60 nm ¼ d
|
/2 and 1.74 nm ¼ d
|
/3
(Fig. 6, bottom). From these data an approximation of
the molecular shape of lipid A is possible: In the absence
of HDL, it is conical with a higher cross-section of the
hydrophobic than the hydrophilic moiety, and is conver-
ted into a cylindrical one in the presence of HDL.
HDL secondary structure
The secondary structure of the apolipoprotein (apoA-I)
part of HDL was determined by IR-spectroscopy by
analyzing the amide I-vibration (predominantly C¼O
stretching vibration) in the spectral range 1700–1600 cm
)1
in H
2
O-containing as well as D
2
O-containing buffer. IR
spectra are given in the range 1700–1400 cm
)1
at a [LPS]/
[HDL] ratio of 1 : 0.5 weight percentage in D
2
O(Fig.7A)
exhibiting the amide I¢-vibration centered around

contour except for the fact that the peak position of the
amide I vibration is shifted to approximately 1658 cm
)1
.
Zeta potential
The zeta potential as an indicator for accessible surface
charges was determined for LPS Re and lipid A in the
presence of increasing amounts of HDL. From Fig. 8 it can
be deduced that the pure endotoxins have a high negative
surface charge corresponding to potential values of )50 to
Fig. 5. Peak position of the symmetric stretching vibration of the
methylene groups m
s
(CH
2
) vs. temperature for a 10-m
M
dephospho-LPS
Re preparation at different HDL concentrations.
Fig. 6. Synchrotron radiation X-ray diffraction patterns of lipid A (top)
and a mixture of lipid A and HDL (bottom, weight ratio 1 : 0.5) at 90%
water content. The diffraction pattern of the aggregate structure of lipid
A indicates the existence of a superposition of a unilamellar with a
cubic inverted structure, that of the mixture a multilamellar structure.
Ó FEBS 2002 HDL interaction with endotoxins (Eur. J. Biochem. 269) 5977
)60 mV, which is increasingly compensated by the addition
of higher amounts of HDL. However, the charge compen-
sation seems to be completed at a weight ratio [endotoxin]/
[HDL] 1 : 1 at a remaining potential of )20 mV. Therefore,
HDL does not compensate the negative charges of the

transport of LPS into PtdSer as example of negatively
charged phospholipids was determined by fluorescence
resonance energy transfer spectroscopy in the absence and
presence of HDL (Fig. 9). The addition of LPS at t ¼ 50 s
indicates that LPS itself does not intercalate into the PtdSer
liposomes, the following addition of LBP at t ¼ 150 s leads
to an rapid increase in NBD-fluorescence intensity corres-
ponding to the LBP-mediated intercalation of LPS and LBP
into the PtdSer liposomes (Fig. 9A). The addition of HDL
at t ¼ 50 s leads to an increase in the NBD-fluorescence
intensity indicating an intercalation of HDL into the PtdSer
liposomes, the subsequent addition of LPS at t ¼ 100 s
apparently leads to an HDL-mediated transport of LPS
into the target cell membrane (Fig. 9B), as the addition of
pure buffer instead of LPS at this time causes a reduction of
the fluorescence intensity due to dilution (data not shown).
The final addition of LBP at t ¼ 150 s leads to another
increase in the NBD-fluorescence intensity caused by
intercalation of pure LBP and LBP-mediated intercalation
of LPS into the PtdSer liposomes (Fig. 9B). In Fig. 9C, the
addition of LPS first and then of HDL again showed no
intercalation of LPS by itself, an intercalation of HDL as
found already in Fig. 9(B), and the final strong increase of
the NBD-fluorescence intensity indicates the intercalation of
LBP and the [LPS]–[LBP] complex. In Fig. 9D, after
addition of the preincubated complex (LPS + HDL) the
increase of the NBD-fluorescence intensity indicates an
intercalation of HDL and (LPS + HDL) complex, which
is followed by the strong increase due to LBP-induced
intercalation.

Fig. 8. Zeta potential of 0.5 m
M
lipid A and LPS Re preparations
in dependence on different [endotoxin]/[HDL] weight ratios from the
determination of the electrophoretic mobility by laser Doppler
anemometry.
5978 K. Brandenburg et al. (Eur. J. Biochem. 269) Ó FEBS 2002
held constant (10 ngÆmL
)1
) while the HDL concentration
was increased (10 ngÆmL
)1
,100ngÆmL
)1
,1lgÆmL
)1
,
10 lgÆmL
)1
) to produce the weight ratios shown. As plotted
in Fig. 10, three types of experiments with LPS Re and
HDL have been carried out. Preincubation of the cells
withLPSRe(30minat37°C) and following addition of
HDL, preincubation of the cells with HDL and following
addition of LPS Re, and the incubation of the cells with
(LPS + HDL) complexes. Preincubation of the cells with
HDL and following addition of LPS Re, and the (LPS +
HDL) complex leads in all examined concentrations to a
decrease in IL-6 production. Preincubation of the cells with
LPS Re and following addition of HDL leads to an

which manifests in a binding to the lipid A backbone, in
particular to the diglucosamine-phosphate region (Fig. 1),
to an increase of the phase transition temperature of the acyl
chains of the endotoxins and a drastic increase in acyl chain
order, i.e. a rigidification of the endotoxin aggregates
(Fig. 2). As the m
s
(CH
2
) signal results from both, the acyl
chains of LPS and of the phospholipids from the HDL
particles, a pure addition of the signals would lead to a curve
somehow in between those of pure HDL and pure LPS, i.e.
it would indicate (Fig. 2) a Ôfluidization of LPSÕ.The
observation of the rigidification therefore can be assumed to
be even stronger than found in Fig. 2 due to the superpo-
sitions of the HDL and phospholipid signals. The phase
transition enthalpy of LPS with DH ¼ 38 kJÆmol
)1
is
slightly larger compared with the data reported for phos-
pholipids [10], but considering the difference in the number
of acyl chains (six for LPS instead of two for phospholipids),
it is strongly reduced for LPS as compared with saturated
phospholipids. The decrease of the phase transition
Fig. 10. LPS-induced IL-6 production of human mononuclear cells by
10 ngÆmL
)1
LPS Re and at different [LPS]/[HDL] weight ratios was
determined in three types of examinations. Preincubation of the cells

in the gel phase below T
c
aremoreorlessthesame.
Beside the lipid A phosphate groups as target structures
(Figs 1, 4, 5 and 8) the change of the phase transition of
dephospho LPS (Fig. 4) and the remaining zeta potential
after binding saturation (Fig. 8) give a hint that HDL
binds also to other target structures in the endotoxins, for
example to the sugar part of the endotoxins as deduced
from the band intensity decrease of the diglucosamine ring
mode (Fig. 1). This interpretation is strongly supported by
the biological data: Coincubated (LPS + HDL) com-
plexes lead in both test systems to a significant decrease of
the signals (IL-6 production and LAL coagulating acti-
vity). It has been reported for synthetic endotoxins that
LAL activity is highest for preparations with a digluco-
samine backbone including the 4¢-phosphate (compound
504), whereas the sample without 4¢-phosphate but with
1-phosphate (compound 505) was less active by one order
of magnitude [23]. Thus, the binding of HDL to LPS
must comprise at least the diglucosamine backbone
inclusive the 4¢-phosphate (see also Fig. 1) which inhibits
the activity in the LAL at all concentrations (Fig. 11). In
previous papers, we have reported that binding of various
proteins (hemoglobin, lactoferrin, recombinant human
serum albumin) lead to systematic changes (increase or
decrease in dependence on the protein) in cytokine
induction, but there was no corresponding behavior in
the LAL test [9,15,21]. This can now be interpreted as
resulting from different target structures (epitopes) of the

completely different characteristic: Albumin (in its recom-
binant form) compensates the phosphate charges to an only
very low degree, the zeta potential remains lower than
)40 mV, which seems to be connected with the observation
that albumin does not reduce the immunostimulatory
activity of LPS, rather a slight increase is observed [9].
Together with the data for hemoglobin, for which also no
binding to the phosphate groups of LPS and no reduction of
the immunostimulatory activity can be found [21], it may be
hypothesized that a basic prerequisite for a decrease of the
endotoxicity of LPS is the neutralization of the its negative
charges.
The binding process of HDL to the endotoxins is
accompanied by a dramatic decrease of the LPS immu-
nostimulatory activity which is strongest when HDL is
added before LPS to the cells (Fig. 10). One possible
explanation is the change of the aggregate structure from
a mixed unilamellar/cubic into a multilamellar one
(Fig. 6). In the former structures, the binding structures
(epitopes) may be accessible to proteins. Within the
multilamellar stacks, in contrast, the epitopes of the
endotoxins are more or less hidden, thus leading to a
considerable decrease of interacting molecules such as
LBP, soluble (s) or membrane-bound (m) CD14 (sCD14
and mCD14), or other receptor proteins on the cell
surface [25–30]. Another pathway, however, is also
probable. HDL by itself incorporates into phospholipid
liposomes (Fig. 9B,C) which is also valid for the [LPS]–
[HDL] complex (Fig. 9D), that means there is some
similarity to the action of LBP [17]. After incorporation of

This work was financially supported by the Deutsche Forschungsg-
emeinschaft (projects Br 1070/3–1 and SFB 367/B8).
5980 K. Brandenburg et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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