Báo cáo Y học: Biophysical characterization of the interaction of high-density lipoprotein (HDL) with endotoxins doc

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

exhibited an increase in the gel to liquid crystalline phase
transition temperature T
c
and a rigidification of the acyl
chains of the endotoxins as measured by Fourier-transform
infrared spectroscopy and differential scanning calorimetry.
The functional groups of the endotoxins interacting with
HDL are the phosphates and the diglucosamine backbone.
The finding of phosphates as target groups is in accordance
to measurements of the electrophoretic mobility showing
that the zeta potential decreases from )50 to )60 mV to
)20 mV at binding saturation. The importance of the sugar
backbone as further target structure is in accordance with the
remaining negative potential and competition experiments
with polymyxin B (PMB) and phase transition data of the
system PMB/dephosphorylated LPS. Furthermore, endo-
toxin binding to HDL influences the secondary structure of
the latter manifesting in a change from a mixed a-helical/
b-sheet structure to a predominantly a-helical structure. The
aggregate structure of the lipid A moiety of the endotoxins as
determined by small-angle X-ray scattering shows a change
of a unilamellar/inverted cubic into a multilamellar structure
in the presence of HDL. Fluorescence resonance energy
transfer data indicate an intercalation of pure HDL, and of
[LPS]–[HDL] complexes into phospholipid liposomes. Fur-
thermore, HDL may enhance the lipopolysaccharide-bind-
ing protein-induced intercalation of LPS into phospholipid
liposomes. Parallel to these observations, the LPS-induced
cytokine production of human mononuclear cells and the
reactivity in the Limulus test are strongly reduced by the

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

plays a role in binding and neutralizing bacterial
lipopolysaccharide and decrease the immunostimulatory
action of LPS. In particular, a drastic reduction of the
LPS-induced cytokine production [tumor necrosis factor-
a, interleukin (IL)-1, IL-6] due to HDL binding was
observed [2–4]. Furthermore, it was demonstrated that
lipopolysaccharide-binding protein (LBP) increased the
uptake of LPS by reconstituted HDL (R-HDL) particles
derived from either LPS ÔmicellesÕ or LPS–sCD14 com-
plexes, and in this process LPS molecules are exchanged
with phospholipids [5].
Here, we report on the interaction of HDL with deep
rough mutant LPS Re and the Ôendotoxic principleÕ, lipid
A applying a variety of physical and biological techniques.
With Fourier-transform infrared spectroscopy (FTIR)
the phase transition behavior of the acyl chains of the
Correspondence to K. Brandenburg, Forschungszentrum Borstel,
Biophysik, Parkallee 10, D-23845 Borstel, Germany.
Fax: +49 4537 188632, Tel.: + 49 4537 188235,
E-mail:
Abbreviations: ATR, attenuated total reflectance; FTIR, Fourier-
transform infrared spectroscopy; HDL, high-density lipoprotein;
IL, interleukin; LAL, Limulus amebocyte lysate; LBP, lipo-
polysaccharide-binding protein; LPS, lipopolysaccharide; PMB,
polymyxin B; PtdSer, phosphatidylserine.
(Received 2 September 2002, revised 18 October 2002,
accepted 24 October 2002)
Eur. J. Biochem. 269, 5972–5981 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03333.x
endotoxins in absence and presence of HDL as well as
the effect of HDL on functional groups of the endotoxins

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/

chloroform/petrol ether method [6] from bacteria grown at
37 °C, purified, and lyophilized. Free lipid A was isolated by
acetate buffer treatment of LPS R595. After isolation, the
resulting lipid A was purified and converted to its triethyl-
amine salt.
The known chemical structure of lipid A from LPS R595
was checked by the analysis of the amount of glucosamine,
total and organic phosphate, and the distribution of the fatty
acid residues applying standard procedures. The amount
of 2-keto-3-deoxyoctonate never exceeded 5 weight %.
Dephospho-LPS Re was prepared from LPS deep rough
mutant F515 from Escherichia coli byHFtreatmentatlow
temperature (4 °C). The detailed procedure is described
elsewhere [7].
High-density lipoprotein (HDL) from human plasma was
purchased from Fluka (Deisenhofen, Germany). It was
essentially free of contaminants, in particular of LPS, which
was examined by applying the Limulus test (see later).
Lipopolysaccharide-binding protein (LBP) was a kind
gift of S. F. Carroll (XOMA corporation, Berkeley, CA,
USA).
Sample preparation
The lipid samples were usually prepared as aqueous disper-
sions at high buffer content, i.e. above 60% using 20 m
M
Hepes (pH 7). For this, the lipids were suspended directly in
buffer, sonicated and temperature-cycled several times
between5and70°C and then stored for at least 12 h
before measurement. For the elucidation of the protein
secondary structure in the absence and presence of endo-

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

strong water bands.
In the case of overlapping bands, in particular for the
analysis of amide I-vibration mode, curve fitting was
applied using a modified version of the
CURFIT
program
obtained by D. Moffat, NRC, Ottawa, Canada. An
estimate of the number of band components was obtained
from deconvolution of the spectra [8] and the curve was
fitted to the original spectra after subtraction of base lines
resulting from neighboring bands. The bandshapes of the
single components are superpositions of Gaussian and
Lorentzian. Best fits were obtained by assuming a Gauss
fraction of 0.55–0.60. The precision of the curve fit
procedure is approximately 3%.
ATR
The lipids were prepared as oriented thin multilayers as
described previously [9] by spreading a 1-m
M
lipid suspen-
sion, which was temperature-cycled between 5 and 70 °C
several times prior to spreading, in Hepes buffer on a ZnSe
ATR crystal and evaporating the excess water by slow
periodic movement under a nitrogen stream at room
temperature. The lipid sample was placed in a closed
cuvette, and the air above the sample was saturated with
water vapor to maintain full hydration. Infrared ATR
spectra were recorded with a mercury–cadmium–telluride
detector with a scan number of 1000 at a resolution of
2cm

)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

(s ¼ 2sinhÆk
)1
,2h
scattering angle and k the wavelength ¼ 0.15 nm) were
recorded at 40 °C with exposure times of 2 or 3 min using a
linear detector with delay line readout [12]. The s-axis was
calibrated with tripalmitate, which has a periodicity of
4.06 nm at room temperature. Details of the data acquisi-
tion and evaluation system can be found elsewhere [13]. The
diffraction patterns were evaluated as described previously
[14] assigning the spacing ratios of the main scattering
maxima to defined 3D structures. The lamellar and cubic
structures are most relevant here. They are characterized by
the following features: (a) lamellar: The reflections are
grouped in equidistant ratios, i.e. 1, 1/2, 1/3, 1/4, etc. of the
lamellar repeat distance dL; (b) cubic: The different space
groups of these nonlamellar 3D structures differ in the
ratio of their spacing. The relation between reciprocal
spacing s
hkl
¼ 1/d
hkl
and lattice constant a is s
hkl
¼
[(h
2
+k
2
+l

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-

ment, both after thorough degassing of the suspensions.
After temperature equilibration, the HDL was titrated in
5 lL portions every 10 min into the endotoxin-containing
cell, and the heat for each injection measured by the ITC
instrument was plotted vs. time. The total heat signal from
each experiment was subsequently determined by integra-
ting the individual peaks and plotted against the [HDL]/
[endotoxin] weight ratio.
Fluorescence resonance energy transfer
The fluorescence resonance energy transfer assay was per-
formed as described earlier [16,17]. Briefly, phospholipid
liposomes from phosphatidylserine (PtdSer) were doubly
labeled with the fluorescent dyes N-(7-nitrobenz-2-oxa-1,3-
diazol-4yl)-phosphatidylethanolamine and N-(lissamine
rhodamine B sulfonyl)-phosphatidylethanolamine (Rh-PE)
(Molecular Probes, Eugene, OR, USA). Intercalation of
unlabeled molecules into the doubly labeled liposomes leads
to probe dilution and thus inducing a lower fluorescence
resonance energy transfer efficiency: the emission intensity
of the donor increases and that of the acceptor decreases
(for clarity, only the quotient of the donor and acceptor
emission intensity is shown here).
In all experiments, doubly labeled PtdSer liposomes were
prepared and after 50, 100, and 150 s recombinant LBP,
LPS, and HDL were added in different order, and the NBD
donor fluorescence intensity at 531 nm was monitored for at
least 300 s. LBP, HDL and LPS were added in the weight
ratios 0.5 : 1 : 1.
Stimulation of human mononuclear cells by LPS Re
For an examination of the cytokine-inducing capacity of the

[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

)1
)
were transferred into 96-well culture plates. The stimuli were
seriallydilutedinserum-freeRPMI1640andaddedtothe
cultures at 20 lL per well. The cultures were incubated for 4
hat37 °C under 5% CO
2
. Supernatants were collected after
centrifugation of the culture plates for 10 min at 400 g and
stored at )20 °C until determination of cytokine content.
Immunological determination of IL-6 in the cell super-
natant was performed in a sandwich-ELISA as described
elsewhere [18]. Ninety-six-well plates (Greiner, Solingen,
Germany) were coated with a monoclonal (mouse) human
IL-6 antibody (clone 16 from Intex AG, Switzerland). Cell
culture supernatants and the standard (recombinant human
IL-6, Intex) were diluted with buffer. After exposure to
appropriately diluted test samples and serial dilutions of
standard rIL-6, the plates were exposed to peroxidase-
conjugated (sheep) anti-human IL-6 antibody. The plates
were shaken 16–24 h at room temperature (21–24 °C) and
washed six times in distilled water to remove the antibodies.
Subsequently the color reaction was started by addition
of tetramethylbenzidine/H
2
O
2
in alcoholic solution and
stopped after 5–15 min by addition of 0.5 molÆL
)1

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

units EUÆmL
)1
, and the resolution limit is £ 0.1 EUÆmL
)1
(maximum value for ultrapure water from embryo-transfer,
Sigma).
RESULTS
Measurements of hydrated LPS–HDL complexes
Infrared-ATR experiments were performed with hydrated
LPS multilayers in the absence and presence of different
HDL concentrations. In these measurements, the LPS
concentration was held constant and the spectra were
normalized by taking the band intensity of the symmetric
stretching vibration m
s
(CH
2
) as standard. In Fig. 1, a change
in the band contours in the range of the two phosphate and
the diglucosamine vibrations, m
as
(PO
2
) 1270–1250 cm
)1
,
and m
as
(PO
2

)
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

of the symmetric stretching vibration m
s
(CH
2
), which is a
measure of acyl chain order. HDL induces a slight
rigidification in particular in the liquid crystalline (a) phase
of the acyl chains of LPS Re, as deduced from a decrease in
wavenumber values at a given temperature, and a significant
increase in the phase transition T
c
from 31 °C for pure LPS
to 40 °C for an [LPS]–[HDL] mixture at a weight ratio of
1 : 4. Also, pure HDL exhibits a signal in this wavenumber
range due to its phospholipid moiety. This, however, is
much higher with an only weak temperature dependence in
the wavenumber range 2852.5–2853.5 cm
)1
(Fig. 2). These
values are indicative of acyl chains with a large amount of
gauche conformers. Importantly, the interaction of HDL
with LPS leads to a reduction of the wavenumber by more
than one unit (see vertical line at 37 °C), i.e. a strong
rigidification of the lipid A acyl chains.
This holds true also for lipid A even although higher
amounts of HDL are required to induce a significant
increase in T
c
. Thus, at a weight ratio[lipid A]/[HDL] 1 : 3
the phase transition at T

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

(CH
2
) vs. temperature for a 10-m
M
LPS Re pre-
paration at different HDL concentrations. In the gel (b) phase of the acyl
chains, the peak position lies at 2850 cm
)1
, in the liquid crystalline (a)
phase at 2852.5 cm
)1
.
Ó FEBS 2002 HDL interaction with endotoxins (Eur. J. Biochem. 269) 5975
The phase transition in the first heating scan is characterized
by a coexistence region between 22 and 37 °C(T
1/2
¼
4.5 °C) and the maximum of the heat capacity curve is
found at 31 °CwithDH
C
¼ 38 kJÆmol
)1
. The succeeding
cooling scan reveals only a very small hysteresis for the
re-crystallization of the acyl chains from the liquid crystal-
line to the gel phase. The maximum of the heat capacity
curve of the 1st cooling scan is observed at T ¼ 28 °Cwith
DH ¼ )39 kJÆmol
)1
. A shoulder at  23 °C is observed in

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-

tion ratios. A second peak with a very small enthalpy
contribution at higher temperature (T  63 °C, DH ¼
 8kJÆmol
)1)
corresponds to the denaturation peak of
pure HDL, because the maximum of the heat capacity curve
of pure HDL is observed at  63 °C (Fig. 3C). Thus,
additional HDL does not interact with the LPS membrane
but acts like pure protein. Heating of the sample above
 70 °C leads to complete and irreversible denaturation of
HDL (data not shown).
Parallel to the measurements of LPS Re, differential
scanning calorimetry measurements of the phase behavior
of lipid A indicated a similar increase in T
c
,andthe
evaluation of the phase transition enthalpy (peak area)
showed a value of 14 kJÆmol
)1
which in the presence of
HDL is reduced to 12 kJÆmol
)1
, i.e. a reduction by 15%.
These data indicate that the binding of HDL to LPS and
lipid A leads to a disturbance of the hydrophobic moiety.
Inhibition experiments were performed with the polycat-
ionic peptide polymyxin B (PMB), which binds strongly to
the lipid A phosphates [20]. At a [LPS]/[PMB] weight ratio
of 1 : 0.24, PMB alone causes a drastic fluidization of LPS,
while HDL leads to a rigidification of LPS at a weight ratio

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

) vs. temperature in competition experiments
with LPS Re, PMB and HDL in different sequences.
5976 K. Brandenburg et al. (Eur. J. Biochem. 269) Ó FEBS 2002
weight ratio [dephospho-LPS]/[HDL] 1 : 4.5. As a control,
the effect of PMB on dephospho-LPS Re was monitored. It
is found that PMB causes a slight decrease in T
c
, but no
change in fluidity takes place (data not shown). Therefore,
the phosphates can be assumed not to be the only binding-
sites for HDL.
LPS and lipid A aggregate structures
Synchrotron radiation X-ray small-angle diffraction was
performed with lipid A at 40 °C and at different
concentrations of HDL. The diffraction patterns of pure
lipid A (Fig. 6, top) are indicative of a superposition of a
unilamellar with a cubic inverted structure in accordance
to former results [21], which can be deduced from the
occurrence of the broad interference maximum between
0.1 and 0.4 nm
)1
superimposed by diffraction maxima
at 8.20 nm ¼ 18.4 nmÆÖ5, 5.31 nm ¼ 18.4 nmÆÖ12,
4.08 nm ¼ 18.4 nmÆÖ20 of a periodicity at a
Q
¼
(18.3 ± 0.3) nm (the latter is expressed only very
weakly). In the presence of HDL, this mixed structure
converts into a multilamellar one, which can be deduced
from the occurrence of reflections at equidistant ratios,

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

1653 cm
)1
, but only a very weak amide II-vibration due
to H/D exchange [22]. The evaluation of the amide I¢
vibrational band shows that for HDL in the presence of
Re-LPS ([HDL]/[Re-LPS] 1 : 0.5 weight ratio) in Fig. 7B
the b-turn/antiparallel b-sheet components of the protein’s
secondary structure is changed in favor of the a-helical
component (a detailed assignment of the different secon-
dary structures is presented in the legend of Fig. 7). For
pure HDL the a-helical portion is approximately 34%
and for the complexes ([HDL]/[Re-LPS] 1 : 0.5 weight
ratio) approximately 44%. From the broadening of the
1653 cm
)1
band it becomes obvious that a more hetero-
geneous population of a-helical structures emerge as
consequence of binding to LPS. The occurrence of
unordered (random coil) structures, which in D
2
Oare
located in the range 1640–1645 cm
)1
, can be excluded, as
measurements in H
2
O, for which the unordered structures
are found around 1660 cm
)1
, exhibited a similar band

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

endotoxins completely.
Isothermal titration calorimetry
With ITC an estimate of the stoichiometry of HDL–LPS
binding can be obtained. For this, a LPS dispersion
(0.25 mgÆmL
)1
) within the calorimeter cell was titrated with
a HDL solution (5 lLof12mgÆmL
)1
every 10 min). The
titration yields a negative enthalpy change DH
c
of the LPS–
HDL binding corresponding to an exothermic reaction
(data not shown). A maximum of DH
c
¼ )14 kJ is
observed at a weight ratio of 1 : 1. At higher [HDL]
contents, the DH
c
values decrease to )6 kJ at weight ratios
[HDL]/[LPS] ¼ 4 : 1–6 : 1, but do not decrease to zero.
Unfortunately, the HDL amounts available did not allow to
realize higher HDL concentrations, i.e. to determine the
saturation of binding, which therefore must be significantly
higher than a weight ratio of [HDL]/[LPS] ¼ 6:1.
Intercalation into phospholipid liposomes
It has been shown that LBP mediates the transport of LPS
into negatively charged liposomes [17] which seems to be an
important step in cell activation. Here, the LBP-mediated

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.

Similar results are obtained when the PtdSer is replaced
by phospholipid liposomes corresponding to the composi-
tion of the macrophage membrane [16], only the effects are
significantly weaker.
IL-6-production in mononuclear cells
IL-6 production in human mononuclear cells induced
by LPS Re (10 ngÆmL
)1
) was investigated at different
HDL concentrations. The concentration of LPS Re was
Fig. 7. Infrared spectra in the range 1700–1400 cm
)1
for a hydrated sample of [LPS]/[HDL] = 1 : 2 weight ratio (A) and in the range of the amide I¢
(predominantly C=O stretch) vibration for hydrated samples of HDL (B, top) and in the presence of Re-LPS ([LPS]/[HDL] = 1 : 2 weight ratio) (B,
bottom). The measurements were performed at 37 °CinD
2
O. Band component assignments: 1653 cm
)1
, a-helix; 1636–1638 cm
)1
, b-sheet; 1667–
1671 cm
)1
, b-turns; 1682–1685 cm
)1
, b turns and antiparallel b sheet. In the figures, the values of the peak positions and the respective bandwidths
(determinedathalfheight,incm
)1
) are listed. The curve fitting was performed by assuming a Gaussian fraction of 0.6 (Lorentzian fraction 0.4). The
precision of the secondary structural determination is approximately 3%, obtained from repeated measurements (n ¼ 5).

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

insignificant increase in IL-6 production at the lowest HDL
concentration ([LPS]/[HDL] 1 : 1 weight ratio), but at
higher concentrations of HDL also to a decrease in the IL-6
production, but less as compared with the results in the
other experiments.
Biological activity in the LAL assay
The ability of the LPS–HDL complexes to activate the LAL
clotting cascade was measured at LPS Re concentrations of
100 pgÆmL
)1
and 1 ngÆmL
)1
. As shown in Fig. 11 we have
found that HDL reduces the enzymatic activity induced by
pure LPS Re at all investigated concentrations. For
example, at 1 ngÆmL
)1
the activity of 45 EUÆmL
)1
for the
pure LPS Re is reduced to values in the range 15–25
EUÆmL
)1
at all concentrations, starting from [LPS]/[HDL]
1 : 1–1 : 1000 (Fig. 11B). Also, pure HDL was found to be
endotoxin-free as deduced from the low values in the LAL
test which nearly correspond to the values of pure water.
DISCUSSION
The results from the biophysical measurements of the
binding of HDL to endotoxins indicate a strong interaction,

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

with LPS Re (30 min at 37 °C) and following addition of HDL, pre-
incubation of the cells with HDL and subsequent addition of LPS Re,
and incubation of the cells with [LPS Re]–[HDL] complexes.
Fig. 11. Endotoxin activity in the chromogenic Limulus amebocyte
lysate assay at two LPS Re concentrations (100 pgÆmL
)1
and
1ngÆmL
)1
) and different HDL weight ratios.
Fig. 9. Quotient of the fluorescence intensity at 531 nm of doubly
labeled liposomes from PtdSer vs. time. After incubation with LPS Re
and subsequent addition of LBP (A), after incubation with HDL and
subsequent addition of LPS Re and LBP (B), after incubation with
LPS Re and subsequent addition of HDL and LBP (C), and after
incubation with a preincubated mixture of LPS and HDL and sub-
sequent addition of LBP (D).
Ó FEBS 2002 HDL interaction with endotoxins (Eur. J. Biochem. 269) 5979
enthalpy by approximately 22% at a weight ratio of [LPS]/
[HDL] 1 : 1 (Fig. 3) indicates the significant influence of the
acyl chain moiety of LPS in the interaction which might be
connected with the observation that the acyl chain melting
in the presence of HDL does not take place completely. This
may be taken from the wavenumber values of the LPS–
HDL sample above T
c
(Fig. 5) which are by 0.3 cm
)1
lower
than the pure LPS sample, whereas the wavenumber values

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

proteins as found here for HDL.
Concomitant with the binding of HDL to the endotoxins,
a reorientation of the lipid A aggregate structure from
inverted cubic [21] to a multilamellar one (Fig. 6), and a
slight change of the secondary structure of HDL from a
mixed a-helical/b-sheet to a predominantly a-helical struc-
ture (Fig. 7) take place. From this, a model of the LPS–
HDL interaction can be deduced. The binding of HDL
takes place essentially to the diglucosamine sugar backbone
and the 4¢-phosphate of lipid A. The binding-places within
the HDL moiety at present cannot be given. A further
possibility of LPS binding to HDL, an incorporation of the
LPS bilayer into the HDL interior, the phospholipid bilayer
[24], does not seem to be probable as this would not explain
the data for the 1-phosphate and diglucosamine groups as
binding sites as well as the rigidification of the acyl chains.
Still unclear is the binding stoichiometry of the LPS–
HDL system. The data from the zeta potential (Fig. 8)
indicate a value around 1 : 1 weight ratio. At this ratio,
from an estimate of the molecular weights some hundreds
LPS molecules per HDL apolipoprotein can be calculated.
This is, however, far below saturation. Isothermal titration
calorimetric (ITC) experiments showed up to a weight ratio
of 1 : 6 [LPS]/[HDL] still no saturation. This is also in
accordance to the biological data that a high excess weight
ratio of HDL to LPS is necessary for a saturation of the
tumor necrosis factor-a production (Fig. 10).
The comparison of the results of the interaction of LPS
with HDL to those published for the interaction of the
former with another serum protein, albumin, indicates a

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

these molecules into target membranes, a process which is
also enhanced by the action of LBP (Fig. 9A), the
decrease of cell activation may be understood in the light
of our conformational concept [31]. Only those LPS with
a conical shape of their lipid A moiety, corresponding to
an inverted (cubic, H
II
) aggregate structure, represent a
sufficiently high sterical stress at the site of a signaling
protein such as the ion channel Maxi K [32] to induce cell
signaling and, with that, cytokine induction. LPS with a
lipid A moiety having a cylindrical shape are not able to
induce this stress. They are therefore agonistically inactive,
but may block the action of active endotoxins by
occupying the binding-sites [33]. According to this model
and the present data, the reaggregation of the lipid A
moiety due to HDL binding from a cubic into a multi-
lamellar structure would correspond to a change from a
conical into a cylindrical molecular conformation, and
would thus explain the loss of its ability to induce
cytokine production.
ACKNOWLEDGMENTS
We are indebted to C. Hamann, and U. Diemer for performing
fluorescence spectroscopic and Limulus amebocyte lysate measure-
ments, respectively. The expert help of B. Fo
¨
lting for performing the
differential scanning calorimetry experiments is kindly acknowledged.
We thank S.D. Carroll (XOMA Corporation, Berkely, CA, USA) for
the kind gift of LBP.

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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