Biophysical characterization of synthetic rhamnolipids · 1 Biophysical characterization of...

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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-

14195 Berlin, Germany

3European 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

mailto:[email protected]

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Summary

Synthetic rhamnolipids, derived from a natural diacylated glycolipid, RL-2,214, produced by

Burkholderia (Pseudomonas) plantarii, were analysed biophysically. Changes in the chemical

structures comprised variations in the length, the stereochemistry and numbers of the lipid

chains, numbers of rhamnoses, and the occurence of charged or neutral groups. As relevant

biophysical parameters the gel (β) to liquid crystalline (α) phase behaviour of the acyl chains

of the rhamnoses, their three-dimensional supramolecular aggregate structure, and the ability

of the compounds to intercalate into phospholipid liposomes in the absence and presence of

lipopolysaccharide-binding protein (LBP) were monitored. Their biological activities were

examined as the ability to induce cytokines in human mononuclear cells (MNC) and to induce

chemiluminescence in monocytes. Depending on the particular chemical structures, the

physicochemical parameters as well as the biological test systems show large variations. This

relates to the acyl chain fluidity, aggregate structure, and intercalation ability, as well as the

bioactivity. Most importantly, the data extend our conformational concept of endotoxicity,

based on the intercalation of naturally-originating amphiphilic virulence factors into

membranes from immune cells. This ‘endotoxin conformation’, produced by amphiphilic

molecules with hydrophilic charged backbone and apolar hydrophobic moiety and adopting

inverted cubic aggegate structures, causes a strong mechanical stress in target immune cells

on integral proteins eventually leading to cell activation. Furthermore, biologically inactive

rhamnolipids with lamellar aggregate structures antagonize the endotoxin-induced activity in

a way similar to lipid A-derived antagonists.


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Beside the well-known bacterial cell wall components such as lipopolysaccharide (LPS,

endotoxin), peptidoglycan (PG) and lipopeptides, which enable the immune system to

recognize and identify the invading pathogen as “non-self”, also virulence factors secreted as

exotoxins by pore-forming toxins may lead to severe infections in mammals [1]. Furthermore,

it was found in Burkholderia pseudomalei cells that also higher concentrations of

rhamnolipids were present in the biomass [2]. These heat-stable extracellular toxins,

characterized as rhamnolipids, exhibited considerable cytotoxic and hemolytic activity as well

as significant antimicrobial activity [2,3]. Their surfactant activity may play a major role in

the degradation of hydrophobic compounds used in the application of the microorganisms in

bioremediation and biotransformation [4]. Recently, we have characterized a rhamnolipid, 2-

O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-(R)-3-hydroxytetra-decanoyl-(R)-3-hydroxy-

tetradecanoate (RL-2,214) from Burkholderia (Pseudomonas) plantarii biophysically,

including the characterization of the acyl chain melting of the hydrocarbon chains, the type of

structure of rhamnolipid aggregates, their incorporation into target cell membranes, their

critical micellar concentration, their ability to induce biological activity such as cytokines in

human mononuclear cells and the involvement of cell surface receptors in this process, their

ability to activate the Limulus cascade, and their antibacterial activity [5]. The results showed

a structure-activity relationship similar to that of bacterial LPS on the one hand but with a

number of distinct characteristics on the other. To get more general informations on the

dependence of particular functional groups in the structure-activity relationship, we have

synthesized a variety of rhamnolipids based on the structure of RL-2,214. These variations

comprise number of rhamnoses in the polar moiety (1 to 3), number of acyl chains (2 or 3),

stereochemistry (3(R) and 3(S)-OH) and length of the acyl chains (C:4 to C:18), and head

group charge (negatively charged carboxylate or neutral hydroxy group), see Fig. 1. We have

found that the different physicochemical characteristics of the compounds sensitively depend

on the particular chemical structure, and also determine their ability to cause activity in


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immune cells. In this way, suitable designed rhamnolipids fulfill the criteria to act in a similar

way as bacterial endotoxins (lipopolysaccharides, LPS) and thus constitute an ‘endotoxic

conformation’.

Results

Cytokine-inducing activity

The ability of the rhamnolipids to induce cytokine production (TNFα) in human mononuclear

cells (MNC) was tested in comparison with rough mutant lipopolysaccharide (LPS Ra) (Fig.

2). Clearly, LPS Ra causes cytokine induction down to at least 1 ng/ml, whereas the activity

of all rhamnolipids presented in Fig. 2 is more than two orders of magnitude lower, but

significantly present down to 100 ng/ml. In detail, RL 9 corresponding to the natural structure

and that with one additional rhamnose residue (RL4) are the strongest activators.

Rhamnolipids RL2, 3, 5, 10 and 11 not listed in the figure are completely inactive, i.e., the

triacylated compounds (RL2 and 3), that with longer acyl chains (RL5), and the non-charged

compounds (RL10 and 11) do not induce any TNFα even up to 10 µg/ml.

It should be noted that after dilution of the rhamnolipids in RPMI nutrient as well as in

destilled water rather than in buffer all biological activities vanished.

Induction of chemiluminescence in monocytes

The ability of the rhamnolipids to induce reactive oxygen intermediates in monocytes was

tested in comparison to that of LPS Ra. From the time kinetics of the chemiluminescence of

the samples at a concentration of 0.1 µg/ml, the average chemiluminescence rates after 45

minutes were o9btained at three concentrations of 0,1, 1, and 3 µg/ml, respectively (Fig. 3).

Clearly, LPS induces the strongest reaction. The natural compound RLnat and compounds


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RL4 and 9, but also RL7 exhibit significant activity at all concentrations. In particular, except

for RL10, which exhibits a small signal at 3 µg/ml, the rhamnolipids inactive in the cytokine

test are also inactive in this assay.

Fourier transform infrared spectroscopy (FTIR)

For the identification of particular functional groups, infrared spectra of rhamnolipids at 95 %

water concentration were analysed. Vibrational bands around 1410 and 1585 cm-1 for the

rhamnolipids with carboxyl groups were indicative that the hydrated molecules are negatively

charged (data not shown). Furthermore, their spectra exhibit vibrational bands typical for the

hydrophobic region (symmetrical and antisymmetrical) νs and νas stretching vibration of -

CH2- groups around 2920 and 2850 cm-1, respectively. These are sensitive markers of acyl

chain order. In Figure 4, the peak positions of the symmetric stretching vibrational band

νs(CH2), are plotted versus temperature for all investigated rhamnolipids. Clearly, the

temperature course for the natural and synthetic compound RL-2,214 (RLnat and RL9) are

nearly identical, with a phase transition temperature Tc around – 10 °C and a further gradual

increase of the wavenumber at higher temperatures indicating a high fluidity of the acyl

chains (Fig. 4C). RL7 with one rhamnose does not strongly change the picture, whereas RL4

with 3 rhamnoses exhibits a high fluidity at all temperatures (Fig. 4B). The changed

stereospecifity (RL13) is connected with a decrease of Tc to below – 15 °C, but no change of

the fluidity at higher temperatures. The Tc of RL3 with one more acyl chain is increased to –5

°C (Fig. 4B,C), whereas the change of the length of the acyl chains leads to a significant

increase in fluidity for the shorter chain RL8 and a decrease in fluidity and increase in Tc for

the longer chain RL5 (Fig. 4A-C). For compound RL12 with one rhamnose and a changed

stereospecific configuration Tc increases to higher temperatures between –3 and 0 °C (Fig.

4B), and the addition of a third rhamnose (RL2) leads to a Tc of even 15 °C (Fig. 4A).


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The shortening of one of the acyl chains (RL1 and 6) in each case leads to an increase in

fluidity at all temperatures (Fig. 4A). The conversion of the charged compounds RL7 and 9

into a neutral form (RL11 and 10) leads to a sharpening of the phase transition, but not much

to a change of fluidity at higher temperatures.

X-ray diffraction

Small-angle X-ray diffraction was applied for the elucidation of the aggregate structures of

selected rhamnolipids 3, 4, 7, 10, and 13, differing in the ability to induce cytokines, at high

water contents and at different temperatures.

In Fig. 5, small-angle X-ray diffraction patterns are presented for biologically inactive RL3

and active 7 in the temperature range 5 to 60 °C. The patterns of RL3 (Fig. 5A) are nearly

identical at all temperatures and are indicative for a multilamellar structure deduced from the

periodicity at 3.77 nm and the second order reflection at 1.88 nm (Fig. 5B, bottom). The

patterns for biologically active RL7 (Fig. 5B) are much more complex and exhibit non-

lamellar characteristics, i.e., reflections are observed which are not located at equidistant

ratios. Furthermore, the spacing ratios of the reflection at lowest scattering vector, 8.93 nm at

40 °C, is untypical for a lamellar but would rather correspond to a reflection typical for non-

lamellar cubic structures (Fig. 5B, bottom). However, due to the low number of observable

reflections no assignment to a particular cubic phase is possible.

Similarly, compounds RL4 and 10 were analysed. Inactive RL10 has a mainly lamellar

characteristics with a main reflection at 4.78 (5 °C) and 4.57 nm (20 °C) which decreases to

3.47 nm (40 °C) and 3.41 nm (60 °C) (data not shown). In contrast, for active RL4 the

ocurrence of a reflection located at 1/√2 of the periodicity typical for a cubic structure is

observed (not shown).

Fluorescence resonance energy transfer (FRET)


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FRET was applied to test the ability of selected rhamnolipids to intercalate into liposomal

membranes made from negatively charged phosphatidylserine (PS, Fig. 6) in the absence and

presence of lipopolysaccharide-binding protein (LBP). The results of Fig. 6 show a change of

the FRET signal after addition at 50 s for all investigated samples RL7,9,10 and 13 indicating

their incorporation into the target liposomes. The addition of LBP after 100 s leads to another

increase of the FRET signal already for the pure liposomes corresponding to an intercalation

of LBP into PS-liposomes. A stronger increase is observed for the rhamnolipids, indicative for

an LBP-mediated intercalation into the liposomes, with the smallest increase for uncharged

RL10, and higher increase in particular for RL7. All measurements were also performed by

using liposomes corresponding to the composition of the macrophage membrane, as described

in the previous report [5], with phosphatidylcholine as the main component. It turned out that

the rhamnolipids intercalated in a similar way into the liposomes, although with a smaller

amplitude (data not shown).

Involvement of cell surface receptors CD14, TLR2, and TLR4

To investigate a possible involvement of the cell surface receptors CD14, TLR2, and TLR4 in

the recognition of the rhamnolipids, its activity in a Chinese hamster ovary (CHO) cell

reporter system was compared with the activity of bacterial LPS, a bacterial lipopeptide

Pam3CSK4, and interleukin-1 (IL-1). The CHO cells are natural TLR2 knockouts and express

human CD25 surface antigen upon induction of NF-κB translocation. Experiments with

CHO/CD14 alone (expressing only endogenousTLR4) show that they react with LPS, but not

with Pam3CSK4 and RL1,4,7, and 9 (data not shown). The reactivity to cells CHO/CD14-

huTLR2 additionally transfected with human TLR2 showed the expected response to

Pam3CSK4, but again there was no significant increase of the TLR2 signal for all investigated

rhamnolipids (not shown).


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MaxiK channel activity

The ability of the K+- channel (MaxiK) blocker paxilline, which effectively inhibits the

endotoxin-induced cytokine production in macrophages, to influence the TNFα production of

these cells was also tested for RL4, 9, and 13 as compared to control LPS. It was found that

paxilline is able to reduce the rhamnolipid-induced cell activation significantly at

concentrations of 10 and 20 µM at which the LPS-induced activation of cells was blocked

completely (Fig. 7). Of course, it has again to be considered that the activation of

macrophages takes place at a 1000-fold lower concentration of LPS as compared to the

rhamnolipids.

Antagonistic action of inactive rhamnolipids

The rhamnolipids RL2, 3, 5, and 10, which were inactive in the cytokine assay (see above),

were tested with respect to their ability to block the LPS-induced cytokine production in

human mononuclear cells, i.e., to act antagonistically. For this, LPS Re from S.minneota R595

was added to the cells at a concentration of 1 µg/ml and 1 ng/ml, and rhamnolipids were

added at different concentrations with final ratios of [rhamnolipid]:[LPS] 100:1 to 1:1 per

weight. In Fig. 8, the results are given for the RL-2:LPS system. Clearly, at both stock

concentrations the addition of RL-2 to LPS in excess leads to an inhibition of the LPS-

induced TNFα-production, i.e., an antagonistic action takes place. This was found to be

similarly true for the other rhamnolipids RL-3, RL-5, and RL-10 (data not shown).

Discussion

Natural rhamnolipid exotoxins have been described to be antimicrobial, but also cytotoxic to

human cells, in a way of a detergent-like action on target cells [2,4,6]. Also, they have been


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shown to stimulate the uptake of hydrophobic compounds by bacteria [4]. Recently, the

natural rhamnolipid RL-2,214 from Burkholderia plantarii was characterized

physicochemically and with respect to its ability to act as a virulence factor [5]. It has been

found that this rhamnolipid exhibits a variety of endotoxin-related physicochemical

characteristics such as a cubic-inverted aggregate structure, a tendency to intercalate into

target cell membranes, and a suppression of its cytokine induction in mononuclear cells by

polymyxin B due to neutralization of the negative head group charges. A detergent-like

action, however, could not be confirmed, and RL-2,214 also did not show antimicrobial

activity.

For a more general understanding of the underlying mechanisms, we have synthesized a

variety of rhamnolipids differing in the acylation pattern, the number of monosaccharide

residues, and the charge (Fig. 1, Tab. 1). The biological data show clearly, that the active

compounds in two independent test systems (Fig. 2,3) adopt non-lamellar aggregate

structures as exemplarily demonstrated for RL7 (Fig. 5B), whereas the inactive compounds

adopt multilamellar aggregate structures (Fig. 5A). Furthermore, an intercalation into

negatively charged PS liposomes takes place which is enhanced by the action of LBP (Fig. 6).

In contrast, there is apparently no dependence of biological activity on the phase state or the

acyl chain fluidity of the rhamnolipids. For example, inactive RL5 has a relatively high Tc,

whereas for active RL9 it is very low (Fig. 4C). The phase transition behaviour is governed by

rules which also determine the phase transition behaviour of phospholipids [7]. Thus, an

addition of a third acyl chain (RL2) to a diacylated compound (RL7) leads to a drastic

increase of the hydrophobic bulk and, with that, to a considerable increase in Tc from –10 °C

to 15 °C (Fig. 4A,B). Interestingly, the removal of the charge from RL9 leading to RL10 has

– in contrast to the drastic change in bioactivity – nearly no impact on the phase behaviour

(Fig. 4C). Furthermore, the addition of more rhamnoses leads to a fluidization of the acyl

chains (compare mono-rhamnose RL7 with tri-rhamnose RL4, Fig. 4B). This is apparently


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due to the fact that a higher saccharide bulk between neighbouring molecules leads to a looser

packing resulting in more space available for the acyl chains and thus, an increase in fluidity.

Noteworthy is the observation that the rhamnolipids became inactive in pure water (aqua dest)

as well as in RPMI nutrient. The former solvent is of course unphysiological, and we have

observed that also endotoxins loose much activity (one order of magnitude) when diluted in

destilled water (unpublished results). RPMI, in contrast, is such a complex mixture of

different compounds that the reason for the inactivation of the rhamnolipids remains unclear.

These results, on the other hand, confirm the necessity to use physiological buffers in

biophysical experiments.

Regarding the influence of cell activation on the presence of particular receptors, it can be

stated that neither TLR2 nor TLR4 which are important cell membrane receptors for

lipopeptide and lipopolysaccharide structures, respectively [8-10], are recognition structures

for the rhamnolipids (data not shown). In contrast, by adding the specific MaxiK channel

blocker paxilline to macrophages, not only the cell activation by LPS, but also by the active

rhamnolipids is inhibited or at least reduced (Fig. 7). This means that the MaxiK is involved

in the process of signal transduction into the cell interior. It seems obvious that similar as

observed for LPS not only one single receptor but a complete receptor cluster are involved in

cell signalling [11]. Thus, Triantafilou et al. [12] have found that different LPS and LPS part

structures trigger the recruitment of different receptors within microdomains, and the

composition of each receptor cluster seem to determine whether an immune response will be

induced or inhibited.

Independent which membrane proteins are reponsible for cell signalling, apparently one

general principle governs the process of cell activation by amphiphilic compounds: One main

prerequisite for the induction of bioactivity is the adoption of a non-lamellar, preferentially

cubic structure, and another prerequisite is the incorporation into target cell membranes either

by itself or mediated by LBP. In the membrane, the amphiphilic compounds are present in


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domains, and cause a conformational change of signalling proteins. For LPS, these processes

could be verified as published previously [13]. In particular, the role of LBP and the

intercalation of LPS aggregates into target membranes could be proven [14,15]. The

interpretation that rhamnolipids may assume domains in target cell membranes is supported

by a recent study of Sanchez et al [16], who observed domain formation of a natural

dirhamnolipid in a phosphatidylethanolamine matrix.

Together with the present work for synthetic rhamnolipids we think that the above presented

signalling pathway holds true beside for LPS also for a glycolipid from Mycoplasma

fermentans [16], for synthetic phospholipid-like molecules [17,18] various monophosphoryl

lipid A analogues, and for bacterial lipopeptides (unpublished).

Interesting is the observation of an antagonistic action of those rhamnolipids, which are

themselves agonistically inactive (see Fig. 8). Compounds on the basis of lipid A part

structures such as tetraacyl lipid A - synthetic compound ‘406’ – need, beside a multilamellar

aggregate structure also a negative head group charge to be antagonistic [19]. For the

rhamnolipids, in contrast, this is not necessary, since also RL10 as uncharged compound was

antagonistic similar as the other compounds RL2, 3, and 5. As stated earlier, the necessity for

the lipid A-like compounds to have a negative charge results from the fact that they are not

able to intercalate into target cell membranes by themselves, a transport protein such as LBP

is needed [20]. For the rhamnolipids, this is apparently not necessary since they can

intercalate by themselves into target cell membranes (Fig. 6) independently of the presence of

a negative net charge.

Experimental Procedures

Synthesis


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The efficient parallel synthesis of rhamnolipids was accomplished by means of the previously

developed concept of Hydrophobically Assisted Switching Phase (HASP) synthesis [21].

Rhamnolipids bearing a di-/trilipid moiety allowed to easily adapt the HASP-method of

flexible switching between solution phase steps and solid-supported reactions on a reversed

phase hydrophobic silica support (bulk RP18) to the assembly of a rhamnolipid library. The

construction of rhamnolipids based on the lead structure RL-2,214 started from

enantiomerically pure β-hydroxycarboxylic esters and -acids of various lengths C4-C18

obtained by enantioselective catalytic Noyori hydrogenation. Both, the esterification of β-

hydroxycarboxylic acids and –esters, removal of protecting groups and subsequent iterative

glycosylation cycles with the tailormade rhamnose donor 3,4-O-(2,3-dimethoxybutane-2,3-

diyl)-2-O-phenoxyacetyl-α-L-rhamnopyranosyl-trichloroacetimidate [22] were performed as

high-yielding parallel HASP-steps (Fig. 9).

Whereas the final hydrolysis of (3R,3’R) configured rhamnolipid methyl esters was performed

with solid-supported lipase from Candida antarctica which furnished the (3R,3’R)-

rhamnolipid acids in good to excellent yields, (3R,3’S) configured rhamnolipid acids were not

recognized by the enzyme and had to be accessed via a debenzylation-route of the

correspondent (S)-3-HO-benzyl esters. Rhamnolipid alcohols required the use of terminally

reversed esters which were accessed via a (R)-3-(2-methoxy ethoxymethyl) protected lipid

actetate.

Lead compound of the synthesis was RL-2,214 (RL9) corresponding to the structure of the

natural rhamnolipid. Removal of one rhamnose leads to RL-1,214 (RL7), addition of one

rhamnose to RL-3,214 (RL4), change of the stereoisomeric configuration to RL-2,2(R)14,(S)14

(RL13), addition of one acyl chains to RL-2,314 (compound 3), and change of the length of the

acyl chains to RL-2,212 (RL8) and to RL-2,218 (RL5). The compound with one rhamnose

(RL7) was additionally modified by changing the stereospecific configuration RL-1,2(R)14,(S)14

(RL12), by adding one additional acyl chain RL-1,314 (RL2), or by shortening the respective Rhamnosyn_revidiert_.doc

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acyl chains (RL 1 and 6). Finally, the carboxylates of compounds with one and two

rhamnoses (RL7 or 9) were changed into a non-charged hydroxy group ( RL11 and 10).

The chemical structures and the nomenclature used for the single compounds are listed in Fig.

1 and Tab. 1.

Other Reagents

Lipopolysaccharides from the rough mutant Re or Ra from Salmonella minnesota (R595 or

R60), respectively, were extracted by the phenol/chloroform/petrol ether method [23] from

bacteria grown at 37 °C, purified, and lyophilized. The lipopeptide palmitoyl3CSK4 was from

EMC Microcollections (Tübingen, Germany). Lipopolysaccharide-binding protein (LBP) was

a kind gift of Russ L. Dedrick (XOMA Co, Berkeley, Ca, USA) and it was stored at -70°C as

a 1 mg/mL stock solution in 10 mM Hepes, pH 7.5, 150 mM NaCl, 0.002% (v/v) Tween 80,

0.1% F68. Bovine brain 3-sn-phosphatidylserine (PS), egg 3-sn-phosphatidylcholine (PC), 3-

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).


18

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.


19

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enhancement of LPS-induced activation of mononuclear cells. Infect Immun 69, 6942-6950.

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21

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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


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

Fig. 1.


23

RL No. M Compound [g/mol]

1 476,60 R-4-14 2 843,22 R-14-14-14 3 989,36 R-R-14-14-14 4 909,15 R-R-R-14-14 5 875,22 R-R-18-18 6 476,60 R-14-4 7 616,87 R-14-14 8 706,90 R-R-12-12 9 763,01 R-R-14-14

10 749,0 R-R-14-14-OH 11 602,90 R-14-14-OH

12 616,9 R-14-(S)14 13 763,01 R-R-14-(S)14

Table 1: Schematic listing of the rhamnolipids investigated.


24

Fig. 1


25

R60 RL1 RL4 RL6 RL7 RL8 RL9 RL12 RL130

200

400

600

800

1000

1200

1400

1600

LPS R60

100 ng/ml 10 ng/ml 1 ng/ml

Fig.2

Con

cent

ratio

n TN

Fα (p

g/m

l) Rhamnolipids

10 µg/ml 1 µg/ml 100 ng/ml


26

Fig. 3


27

-20 -10 0 10 20 30 40 502849

2850

2851

2852

2853

2854C

RL nat RL 9 RL 5 RL 3 RL 10

Temperature (°C)

-10 0 10 20 302850

2851

2852

2853

2854

RL 3 RL 8 RL 4 RL 7 RL 13 RL 12W

aven

umbe

r (cm

-1) -10 0 10 20 30

2850

2851

2852

2853

2854

B

A

RL 6RL 11 RL 1RL 8RL 2

Fig. 4


28

0,1 0,2 0,3 0,4 0,5 0,6 0,7

0,1 0,2 0,3 0,4 0,5 0,6 0,7

5°C 20°C 40°C 60°C

log

I

40 °C

1.88 nm

3.77 nm

log

I

s (nm-1)

Fig. 5A


29

0,1 0,2 0,3 0,4 0,5

0,1 0,2 0,3 0,4 0,5 0,6 0,7

5°C 20°C 40°C 60°C

log

I

4.86 nm

40 °C

2.98 nm

4.46 nm8.93 nm

log

I

s (nm-1)

Fig. 5B


30

0 50 100 150 200 250 300

0,8

1,0

1,2

1,4

1,6

1,8

2,0

2,2RL13RL7RL9

RL10

Buffer/LBP

Buffer/Buffer

+LBP+ Buffer/RL's

FRET

sig

nal (

I D/I A)

Time (s)

Fig. 6


31

RL4 10

µg/m

l

RL4 1µ

g/mL

RL9 10

µg/m

L

RL9 1µ

g/mL

RL13 1

0µg/m

L

RL13 1

µg/m

L

0

100

200

300

400

500

600

700

800

900

1000

[Paxilline]

Con

ccen

tratio

n of

TN

Fα (p

g/m

L)

Samples

0 µM 10 µM 20 µM

Fig. 7


32

0

400

800

1200

1600

2000

0:1100:1

50:120:1

5:11:11:00:1100:1

1:0 50:1 20:1

5:11:1

1 ng/mlLPS Re

1 µg/ml LPS Re

TN

Fα p

rodu

ctio

n of

m

onon

ucle

ar c

ells

(pg/

ml)

[RL2]:[LPS Re] (weight ratio)

Fig. 8


33

Fig. 9


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