1 Biophysical characterization of synthetic rhamnolipids Jörg Howe 1 , Jörg Bauer 2 , Jörg Andrä 1 , Andra B. Schromm 1 , Martin Ernst 1 , Manfred Rössle 3 , Ulrich Zähringer 1 , Jörg Rademann 3 , and Klaus Brandenburg 1* 1 Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Parkallee 1-40, D-23845 Borstel, Germany 2 Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, D-13125 Berlin, Germany and Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, D- 14195 Berlin, Germany 3 European Molecular Biology Laboratory, Outstation Hamburg, EMBL c/o DESY, Notkestr. 85, D-22603 Hamburg, Germany Running title: Biophysics of synthetic rhamnolipids Key words: Rhamnolipids, endotoxins, organic synthesis, cytokine induction, X-ray diffraction * Corresponding author Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Parkallee 10, D-23845 Borstel, Germany Tel: +49 4537-188 235, Fax: +49 4537-188 632, E-mail: [email protected]Rhamnosyn_revidiert_.doc
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Biophysical characterization of synthetic rhamnolipids · 1 Biophysical characterization of synthetic rhamnolipids Jörg Howe1, Jörg Bauer2, Jörg Andrä1, Andra B. Schromm1, Martin
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Biophysical characterization of synthetic rhamnolipids
Jörg Howe1, Jörg Bauer2, Jörg Andrä1, Andra B. Schromm1, Martin Ernst1, Manfred Rössle3,
Ulrich Zähringer1, Jörg Rademann3, and Klaus Brandenburg1*
1Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Parkallee
1-40, D-23845 Borstel, Germany
2Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, D-13125 Berlin,
Germany and Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, D-
sn-phosphatidylethanolamine (PE), and sphingomyelin from bovine brain were from Sigma.
Lipid sample preparation
All lipid samples were prepared as aqueous suspensions in 20 mM Hepes, pH 7. For this, the
lipids were suspended directly in buffer and were temperature-cycled 3 times between 5 and
70 °C and then stored for at least 12 h before measurement. To guarantee physiological
conditions, the water content of the samples was usually around 95 %.
For preparations of liposomes from phosphatidylserine or from a mixture corresponding to the
phospholipid composition of the macrophage membrane (phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, and sphingomyelin in a molar ratio of
1:0.4:0.7:0.5), the lipids were solubilized in chloroform, the solvent was evaporated under a
stream of nitrogen, and the lipids were resuspended in the appropriate volume of buffer and
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treated as described above (temperature-cycling). The resulting liposomes are large and
multilamellar as detected in some electron microscopic experiments (kindly performed by H.
Kühl, Div. of Pathology, Forschungszentrum Borstel).
FTIR spectroscopy
The infrared spectroscopic measurements were performed on an IFS-55 spectrometer (Bruker,
Karlsruhe, Germany). For phase transition measurements, the lipid samples were placed
between CaF2 windows with a 12.5 µm Teflon spacer. Temperature scans were performed
automatically between -20 and 70 °C with a heating rate of 0.6 °C/min. Every 3 °C, 50
interferograms were accumulated, apodized, Fourier-transformed, and converted to
absorbance spectra.
X-ray diffraction
X-ray diffraction measurements were performed at the European Molecular Biology
Laboratory (EMBL) outstation at the Hamburg synchrotron radiation facility HASYLAB
using the SAXS camera X33 [24]. Diffraction patterns in the range of the scattering vector 0.1
< s < 4.5 nm-1 (s = 2 sin θ/λ, 2θ scattering angle and λ the wavelength = 0.15 nm) were
recorded at 40 °C with exposure times of 1 min using an image plate detector with online
readout (MAR345, MarResearch, Norderstedt/Germany). The s-axis was calibrated with Ag-
Behenate which has a periodicity of 58.4 nm. The diffraction patterns were evaluated as
described previously [25] assigning the spacing ratios of the main scattering maxima to
defined three-dimensional structures. The lamellar and cubic structures are most relevant here.
They are characterized by the following features:
(1) Lamellar: The reflections are grouped in equidistant ratios, i.e., 1, 1/2, 1/3, 1/4, etc. of the
lamellar repeat distance dl
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(2) Cubic: The different space groups of these non-lamellar three-dimensional structures
differ in the ratio of their spacings. The relation between reciprocal spacing shkl = 1/dhkl and
lattice constant a is
shkl = [(h2 + k2 + l2) / a ]1/2
(hkl = Miller indices of the corresponding set of plane).
Fluorescence resonance energy transfer spectroscopy (FRET)
Intercalation of the rhamnolipids into liposomes made from phosphatidylserine (PS) alone or
mediated by lipopolysaccharide-binding protein (LBP), was determined by FRET
spectroscopy applied as a probe dilution assay [20]. To the liposomes, which were labelled
with the donor dye NBD-phosphatidylethanolamine (NBD-PE) and acceptor dye Rhodamine-
PE, first the lipids and then LBP, or vice versa, were added, all at a final concentration of 1
µM. Intercalation was monitored as the increase of the ratio of the donor intensity ID at 531
nm to that of the acceptor intensity IA at 593 nm (FRET signal) in dependence on time.
Stimulation of mononuclear cells (MNC)
MNC were isolated from heparinized (20 IE/ml) blood taken from healthy donors and
processed directly by mixing with an equal volume of Hank’s balanced solution and
centrifugation in a Ficoll density gradient for 40 min (21 °C, 500 g). The interphase layer of
mononuclear cells was collected and washed twice in Hank’s medium and once in RPMI
1640 containing 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The
cell number was equilibrated at 5≅106 N/mL. For stimulation, 200 µl/well MNC (1≅106 cells)
were transferred into 96-well culture plates. The stimuli were serially diluted in RPMI-1640
and added to the cultures at 20 µl per well. The cultures were incubated for 4 h at 37 °C under
5% CO2. Cell-free supernatants were collected after centrifugation of the culture plates for 10
min at 400⋅g and stored at –20 °C until determination of the cytokine content.
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Stimulation of macrophages
Monocytes were isolated from peripheral blood taken from healthy donors by the Hypaque-
Ficoll density gradient method. To differentiate the monocytes from the macrophages, cells
were cultivated in Teflon bags in the presence of 2 ng/ml M-CSF in RPMI 1640 medium
(endotoxin < 0.01 EU/ml in Limulus test; Biochrom, Berlin, Germany) containing 2 mM L-
glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, and 4 % heat-inactivated human
serum type AB at 37 °C and 6 % CO2. On day 6 the cells were washed with PBS, detached by
trypsin-EDTA treatment and seeded at 1 ≅ 105/ml in complete medium in 96-well tissue
culture plates (NUNC, Wiesbaden, Germany). After stimulation of the cells with the
rhamnolipidsfor 4 h, cell-free supernatant of duplicate samples were collected, pooled and
stored at –20 °C until determination of cytokine content.
Determination of TNFα concentration
Immunological determination of TNFα in the cell supernatant was performed in a sandwich-
ELISA as described before [5]. 96-well plates (Greiner, Solingen, Germany) were coated with
a monoclonal (mouse) anti-human TNFα antibody (clone 16 from Intex AG, Switzerland).
Cell culture supernatants and the standard (recombinant human TNFα, Intex) were diluted
with buffer. After exposure to appropriately diluted test samples and serial dilutions of
standard rTNFα, the plates were exposed to peroxidase conjugated (sheep) anti-TNFα IgG
antibody. Subsequently, the color reaction was started by addition of
tetramethylbenzidine/H2O2 in alcoholic solution and stopped after 5 to 15 min by addition of
1N sulfuric acid. In the color reaction, the substrate is cleaved enzymatically, and the product
was measured photometrically on an ELISA reader at a wavelength of 450 nm and the values
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were related to the standard. TNFα was determined in duplicate at two different dilutions and
the values were averaged.
To study the influence of the K+-channel (MaxiK) on cytokine induction, the specific channel
blocker paxilline was added at a concentration of 10 and 20 µM 10 min before stimulation by
the rhamnolipids to the mononuclear cells, and incubated at 37 °C.
Chemiluminescence of isolated monocytes
Peripheral blood monocytes were isolated from MNC by counterflow centrifugation
(elutriation) using the JE-6B-elutriator system (Beckman Instruments Inc., Palo Alto, CA,
USA) as described earlier [26]. Monocytes (200.000) were suspended in a modified RPMI-
medium (RPMI-1640-medium without phenol red and sodium bicarbonate but containing 20
mmol /l HEPES [Biochrom, Berlin, Germany]) and the monocytes in a final volume of
200 µl per well were placed in a 96 flat bottom white wells plate (Microlite TCT Flat Bottom
Plate, Dynex Technologies, Inc. Chantilly, VA, USA) . Then the plate was incubated at 37°
for at least 60 min before chemiluminescence measurement. Thereafter the plate was put into
the microplate luminometer MicroLumatPlus (LB 96V, Berthold Technologies, Bad Wildbad,
Germany) and 10 minutes prior to the chemiluminescence measurements luminol (5-amino-
2,3-dihydro-1,4-phthalazinedione, Sigma, Taufkirchen, Germany) was added (10 µl per well
of a 2 mg/ml solution) as the chemiluminescence mediating compound. Then after addition of
2 µl of medium (unstimulated control), of LPS, of natural rhamnolipid compound or
synthetic rhamnolipids, the chemiluminescence of the wells was recorded for 45 minutes
whereby the plate was always kept at 37°C in the luminometer. The data are shown as photon
count rates in relative light units per second (RLU/sec) or as mean RLU per 45 minutes (cf.
Figs. 3).
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Antagonistic action of inactive rhamnolipids
The rhamnolipid samples which were found not to induce any cytokines in human
mononuclear cells were investigated with respect to their ability to block the LPS-induced
TNFα-production in the mononuclear cells. For this, LPS Re from S. minnesota R595 was
prepared at two concentrations 1 µg/ml and 1 ng/ml, and the rhamnolipids were added up to
an exess of 100:1 excess (w/w ).
Activation of CHO reporter cells
The CHO/CD25 reporter cell line, clone 3E10, is a stably transfected CD14-positive CHO
(Chinese hamster ovary) cell line that expresses inducible membrane CD25 (Tac antigen)
under transcriptional control of the human E-selectin promoter pELAM.Tac [27]. It reacts
sensitively to the activation of nuclear factor NF-κB. A TLR2-expressing cell line was
generated by stable transfection of clone 3E10 with human TLR2 (3E10-TLR2).
Acknowledgements
We thank K. Stephan, S. Groth, G. von Busse, and C. Hamann for performing the cytokine
induction assay, the paxilline assay, the infrared, and FRET measurements, respectively. We
thank the Deutsche Forschungsgemeinschaft (SFB 470, project B4 U.Z. and Ra895-3/1 to J.R.
) for financial support, and a stipend from the graduate college ‘Chemie in Interphasen’ to
J.B.
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Figure legends:
Fig. 1: Chemical structures of synthetic rhamnolipid structures.
Fig. 2: Production of TNFα by human mononuclear cells induced by various rhamnolipids in
comparison with LPS from Salmonella minnesota R60. Rhamnolipids RL2,3,5, 10,
and 11 not shown here are completely inactive at all measured concentrations. The
error bars result from the determination of TNFα in triplicate.
Fig. 3a: Kinetics of chemiluminescence in monocytes stimulated with 0.1 µg/ml LPS Re and
various natural (RLnat) and synthetic rhamnolipids.
Fig. 3b: Average chemiluminescent intensity after 45 min for LPS Re and various natural
(RLnat) and synthetic rhamnolipids.
Fig. 4: Gel to liquid crystalline phase behaviour of natural and synthetic rhamnolipids
presented as peak position of the symmetric vibrational band of the methylene groups
versus temperature. In the gel phase of the acyl chains, the peak position is located at
2849 to 2850 cm-1, in the liquid crystalline at 2852.5 to 2853.5 cm-1.
Fig. 5: Synchrotron X-ray diffraction patterns of rhamnolipids RL3 (A) and RL7 (B) in the
temperature range 5-60 °C (top) and at 40 °C (bottom). The scattering vector s = 2 sin
θ/ λ is plotted versus the logarithm of the scattering intensity log I.
Fig. 6: Fluorescence resonance energy transfer spectroscopic (FRET) measurements with
liposomes from phosphatidylserine as FRET signal ID/IA versus time. The
Rhamnosyn_revidiert_.doc
22
rhamnolipids were added at 50 s, and LBP at 100 s to the liposomes. The final
concentrations of liposomes and rhamnolipids were 1 µM, and of LBP 0.1 µM.
Fig. 7: TNFα production of human macrophages induced by rhamnolipids RL4, 9, and 13 at
concentrations 1 and 10 µg/ml and in the presence of the specific MaxiK channel
blocker paxilline at 10 and 20 µM.
Fig. 8: TNFα production of human mononuclear cells induced by two concentrations of LPS
Re (1 µg/ml and 1 ng/ml) in the presence of various concentrations of inactive
rhamnolipid RL2 (antagonistic activity).
Fig. 9: Schematic representation of the chemical synthesis of the rhamnolipids presented in