Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation
Martha Triantafilou1,
Kensuke Miyake2,
Douglas T. Golenbock3,* and
Kathy Triantafilou1,
1 University of Portsmouth, School of Biological Sciences, King Henry Building,
King Henry I Street, Portsmouth, PO1 2DY, UK
2 Department of Immunology, Saga Medical School, Nabeshima, Japan
3 Boston University School of Medicine, Boston Medical Center, The Maxwell
Finland Laboratory for Infectious Diseases, Boston, Massachusetts 02118,
USA
* Present address: Department of Medicine, Division of Infectious Diseases,
University of Massachusetts Medical School, Worcester, MA 01665, USA
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Fig. 1. Receptor molecules implicated in LPS-cellular activation are present in
lipid rafts. MonoMac-6 cells were treated with 1% Triton X-100 buffer for 1
hour on ice and then subjected to sucrose density gradient centrifugation.
Fractions were collected from the top of the gradient; 1%
n-octylglucoside was added to each fraction; and equivalent portions
of each fraction were analysed by SDS-PAGE and immunoblotting. The lipid raft
marker was detected using HRP-conjugated cholera toxin (A), the nitrocellulose
membranes were also probed with 26ic (CD14-specific mAb) (B), hsp70 (C), hsp90
(D), CXCR4 mAbs (E) and GDF5 polyclonal serum (F), as well as with the HTA125
TLR4-specific mAb (G). The relative positions of the raft and non-raft
(soluble) fractions are indicated.
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Fig. 2. Receptor molecules implicated in LPS-cellular activation present in lipid
rafts after LPS stimulation. MonoMac-6 cells were stimulated with 10 ng/ml LPS
in 5% HPS for 30 minutes prior to solubilisation with 1% Triton X-100 buffer
for 1 hour on ice and then subjected to sucrose density gradient
centrifugation. Fractions were collected from the top of the gradient, 1%
n-octylglucoside was added to each fraction, and equivalent portions of each
fraction were analysed by SDS-PAGE and immunoblotting. The lipid raft marker
was detected using HRP-conjugated cholera toxin (A), the nitrocellulose
membranes were also probed with 26ic (CD14-specific mAbs) (B), hsp70 (C),
hsp90 (D), CXCR4 mAbs (E) and GDF5 polyclonal serum (F), as well as with the
HTA125 TLR4-specific mAbs (G). The relative positions of the raft and non-raft
(soluble) fractions are indicated.
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Fig. 3. CD14 and GM-1 ganglioside FRET measurements. Energy transfer between CD14
(FITC-26ic) and GM-1 ganglioside (rhodamine-cholera-toxin) can be detected by
the increase in donor fluorescence after acceptor photobleaching. Donor (FITC)
after (A) acceptor photobleaching and (B) E image. Bar, 5 µm.
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Fig. 4. TLR4 and GM-1 ganglioside FRET mearurements. Energy transfer between TLR4
(FITC-HTA125) and GM-1 ganglioside (rhodamine-cholera-toxin) before (A,B) and
after (C,D) LPS stimulation. Energy transfer can be detected by the increase
in donor fluorescence after acceptor photobleaching. Donor (FITC) after (A,C)
acceptor photobleaching and (B, D) E image. Bar, 10 µm.
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Fig. 5. MCD disrupts lipid raft formation. MonoMac-6 cells were either not treated
(A) or treated (B) with 10 mM MCD for 10 minutes, before solubilisation in 1%
Triton X-100 buffer, followed by raft and non-raft separation by sucrose
density gradient centrifugation. The GM-1 ganglioside distribution was
visualised using HRP-conjugated cholera toxin.
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Fig. 7. Raft-disrupting drugs do not alter surface expression of molecules involved
in LPS-induced cellular activation. MonoMac-6 cells were either mock-treated
(A,C,E) or treated with 10 mM MCD (B,D,F) for 10 minutes before washing with
buffer. Surface expression of CD14 (A,B), CXCR4 (C,D) or TLR4 (E,F) was
determined by flow cytometry. All primary antibodies were visualised with
FITC-conjugated secondary antibodies and analysed by flow cytometry utilising
a FACScalibur (Becton Dickinson) counting 10,000 cells per sample.
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Fig. 8. LPS signalling in lipid rafts. MonoMac-6 cells were either stimulated
(A,C,E) or not stimulated with LPS (B,D,F) prior to treatment with 1% Triton
X-100 buffer for 1 hour on ice and then subjected to sucrose density gradient
centrifugation. Fractions were collected from the top of the gradient, 1%
n-octylglucoside was added to each fraction, and equivalent portions
of each fraction were analysed by SDS-PAGE and immunoblotting. The
nitrocellulose membranes were probed with MyD88 (A,B), Rac-1 (C,D) or SAPK/JNK
(E,F) phospho-specific antibodies followed by incubation of HRP-conjugated
secondary antibodies. The relative positions of the raft and non-raft
(soluble) fractions are indicated.
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© The Company of Biologists Ltd 2002
secretion was measured (D) by treated
monocytes isolated from the blood of healthy donors with 10 mM MCD (black
circles) or 60 µg/ml nystatin (white squares) prior to stimulation with 10
ng/ml LPS in 5% HPS. Control experiments with cells stimulated with LPS in the
absence of raft-disrupting drugs were also performed (eclipse). The effect of
different concentrations of nystatin on LPS-induced TNF-