First published online 20 November 2002
doi: 10.1242/jcs.00212
Lipopolysaccharides from Legionella and Rhizobium stimulate mouse bone marrow granulocytes via Toll-like receptor 2
Robert Girard1,
Thierry Pedron2,
Satoshi Uematsu3,
Viviane Balloy4,
Michel Chignard4,
Shizuo Akira3 and
Richard Chaby5,*
1 Lymphocyte development, URA-1961 of the National Center for Scientific
Research, Pasteur Institute, Paris, France
2 Pathogénie Microbienne Moléculaire, Unité INSERM U389,
Institut Pasteur, Paris, France
3 Department of Host Defense, Research Institute for Microbial Diseases, Osaka
University, Osaka, Japan
4 Défense Innée et Inflammation, Unité associée
IP/Inserm Z485, Institut Pasteur, Paris, France
5 Endotoxin Group, UMR-8619 of the National Center for Scientific Research,
University of Paris-Sud, Orsay, France
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Fig. 1. TLR dependence of cellular responses to the LPS from R. species.
(A) HEK293 cells transfected with the TLR2 and/or TLR6 expression vectors
together with a pELAM luciferase reporter plasmid and pRL-TK were exposed 24
hours later to the Rhizobium species Sin 1 LPS (100 ng/ml) for 8
hours. TLR-dependent activation was measured by the dual-luciferase reporter
assay. (B) BMGs from C3H/HeOU and TLR2-/- mice were incubated with
lipid A fractions (1 µg/ml) isolated from the LPSs of Bordetella
pertussis and Rhizobium species Sin-1. Cell lysates
(105 cells in 1% CHAPS, supplemented with a mixture of protease
inhibitors) were analyzed for CD14 by SDS-PAGE and western blotting.
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Fig. 2. Structures of the lipid A regions of LPSs used in this study. The structure
of the lipid A region of R. species Sin-1 is not completely
determined but, as that from some other Rhizobiaceae
(Bhat et al., 1994 ), it is
devoid of phosphate, has 2-aminogluconate in place of glucosamine-1-phosphate,
and contains the very long chain of 27-hydroxyoctacosanoic acid (27-OHC28:0)
that may be ester-linked in the N-acyloxylacyl residue of the distal
glucosamine unit (Basu et al.,
1999). The lipid A regions of the LPSs from three strains of
Legionella pneumophila
(Zähringer et al., 1995;
Kooistra et al., 2002a) are
built upon the same structural model (two residues of 2,3-diaminoglucose
substituted with one very long and five shorter fatty acids), with variations
limited to the length or substituents of the fatty acid chains in positions 2
and 2': R1=H or OH; R2=OH or CH3;
n=18-22 (in strains CS338 and RC1), or n=16-18 (in strain
811).
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Fig. 4. CD14 expression in BMGs from normal and TLR4-deficient mice. BMGs from
C3H/HeOU, C3H/HeJ and C57BL/10ScCr mice were incubated for 24 hours at
37°C with the B. pertussis LPS, or with LPSs from the different
strains of L. pneumophila. (A) CD14 expression in BMGs incubated with
1 or 10 µg/ml of the LPSs was detected by FACS analysis of the gated
granulocyte population, as described in the legend to
Fig. 3. The dashed line
represents the level of CD14 in non-stimulated cells. (B) CD14 expression in
BMGs incubated with 10 µg/ml of the LPSs was analyzed by western blot in
cell lysates, as described in the legend to
Fig. 1.
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Fig. 5. Analysis of CD14 expression in BMGs from TLR2+/+ and
TLR2-/- mice. BMGs from TLR2+/+ and TLR2-/-
mice were incubated for 24 hours at 37°C with the B. pertussis
LPS, or with LPSs from the different strains of L. pneumophila. (A)
CD14 expression in BMGs incubated with 1 or 10 µg/ml of the LPSs was
detected by FACS analysis of the gated granulocyte population, as described in
the legend to Fig. 3. The
dashed line represents the level of CD14 in non-stimulated cells. (B) CD14
expression in BMGs incubated with 10 µg/ml of re-purified LPSs
(re-extracted with phenol/deoxycholate) was analyzed by western blot in cell
lysates, as described in the legend to Fig.
1.
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Fig. 6. CD14 expression induced by lipid A fractions in BMGs from TLR4- and
TLR2-deficient mice. BMGs from C3H/HeJ, TLR2+/+ and
TLR2-/- mice were incubated for 24 hours at 37°C with lipid A
from B. pertussis (10 µg/ml), or from the different strains of
L. pneumophila (50 µg/ml). CD14 expression was analyzed by western
blot in cell lysates, as described in the legend to
Fig. 1.
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Fig. 7. Analysis of the re-purified lipid A fractions of L. pneumophila
RC1. A re-purified lipid A fraction (re-extracted with phenol/deoxycholate)
from L. pneumophila RC1 analyzed for the presence of contaminants (A)
and for its ability to induce CD14 expression in BMGs from C57BL/10ScCr (B)
and TLR2-/- (C) mice. (A) Samples (40 µg) of LPS from E.
coli J5 and of re-purified lipid A from L. pneumophila RC1 were
analyzed by SDS-PAGE stained with silver nitrate. (B) Western blot analysis of
CD14 expression induced in BMGs from C57BL/10ScCr mice by 10 µg/ml of the
same samples of crude LPS from E. coli and re-purified Lipid A from
L. pneumophila RC1. (C) Western blot analysis of CD14 expression
induced in BMGs from TLR2+/+ and TLR2-/- mice by 10
µg/ml of re-purified LPS and Lipid A from L. pneumophila RC1.
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Fig. 8. Inhibitory effect of B. pertussis lipid A in C3H/HeJ cells. BMGs
from C3H/HeJ mice were preincubated (90 minutes, 37°C) in the presence of
various concentrations of B. pertussis lipid A. (A) The cells were
reincubated (20 hours, 37°C) with (O) or without (•) LPS (1 µg/ml)
from L. pneumophila CS338, and CD14 was detected by FACS analysis of
the gated granulocyte population, as described in the legend to
Fig. 3. (B) The cells were
reincubated (20 hours, 37°C) with or without the lipid A fraction of
L. pneumophila (0.1 µg/ml), and CD14 expression was analysed by
western blot as described in the legend to
Fig. 1.
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© The Company of Biologists Ltd 2003
), RC1
(
), and 811 (
). CD14 was detected by incubation with the
anti-CD14 antibody rmC5-3 (2.5 µg/ml, 30 minutes, 0°C), and further
incubation (30 minutes, 0°C) with a FITC-labeled goat anti-rat Ig mAb. The
percentage of fluorescent cells was determined by FACS analysis of the gated
granulocyte population.