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Cutting Edge: Immune Stimulation by
Neisserial Porins Is Toll-Like Receptor 2 and
MyD88 Dependent
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of June 17, 2017.
Paola Massari, Philipp Henneke, Yu Ho, Eicke Latz, Douglas
T. Golenbock and Lee M. Wetzler
J Immunol 2002; 168:1533-1537; ;
doi: 10.4049/jimmunol.168.4.1533
http://www.jimmunol.org/content/168/4/1533
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References
●
Cutting Edge: Immune Stimulation by Neisserial
Porins Is Toll-Like Receptor 2 and MyD88
Dependent1
Paola Massari,* Philipp Henneke,†‡ Yu Ho,* Eicke Latz,†
Douglas T. Golenbock,† and Lee M. Wetzler2*
N
eisserial porins, the major outer membrane proteins of
the pathogenic neisserial species, are potential antineisserial vaccine candidates and are known to be immunogenic in humans and animals in the absence of exogenous
adjuvants (1, 2). In addition, purified neisserial porins induce an
enhanced immune response against poorly immunogenic Ags (e.g.,
peptides (3)), and a T cell-dependent immune response to T-independent Ags (e.g., capsular polysaccharide (2, 4)). Neisserial porins have been used as adjuvants in various vaccine formulations
such as anti-Haemophilus influenzae type b (5), anti-malaria (3),
anti-pneumococcal polysaccharide conjugate vaccine (6), antimeningococcal polysaccharide conjugate vaccines (7), antimelanoma (8), and anti-group A streptococcus (9).
The mechanism of the adjuvanticity of neisserial porins in vaccine formulations correlates with their ability to up-regulate the
expression of the costimulatory molecule B7-2 (CD86) on the surface of B cells and other APCs (4, 10). The increased surface
expression of B7-2 induces augmented costimulation of T cells
through the interaction with its counterreceptor, CD28 (10). The
*Department of Medicine, Division of Infectious Diseases, Boston University School
of Medicine, Boston, MA 02118; †Department of Medicine, Division of Infectious
Diseases, University of Massachusetts Medical School, Worcester, MA 01665; and
‡
Department of Pediatrics, Free University of Berlin, Berlin, Germany
Received for publication November 2, 2001. Accepted for publication December
21, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
effect of neisserial porins on B cells, including increased B7-2
surface expression and B cell proliferation, is not due to LPS, as
studies were performed using the LPS-nonresponsive mouse strain
C3H/HeJ (4, 10, 11). The lack of response to LPS in these mice is
due to a natural point mutation in the gene encoding the Toll-like
receptor (TLR)34 (12), necessary for LPS-mediated signal transduction (13).
Toll proteins, responsible for the dorsoventral development in
Drosophila (14), are involved in antifungal responses in the adult
fly (15). The mammalian orthologs of Drosophila Toll, termed
TLRs (16), are type I transmembrane proteins belonging to the
pattern recognition receptor family. They are involved in the innate
immune response by recognizing microbial conserved structures
called pathogen-associated molecular patterns (PAMPs) (16), such
as LPS, bacterial lipoprotein, peptidoglycan, lipoteichoic acid,
bacterial unmethylated CpG DNA, mycobacterial lipoarabinomannan, and yeast mannans. The recognition of PAMPs by the pattern
recognition receptor leads to the activation of various intracellular
signaling cascades which modulate nuclear translocation of the
transcription factor NF-␬B (17), induction of cytokines, and expression of effector molecules, such as the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) (18). In this way, it has been
suggested that signaling by the TLRs bridges innate and adaptive
immunity, allowing the host to more efficiently combat microbial
infections.
The best-characterized TLRs, to date, are TLR2 (19) and TLR4
(20). TLR2 is involved in the recognition of Gram-positive bacteria and mycobacteria (21, 22) and bacterial products such as
lipopeptides (23, 24). TLR4 mediates the effect of Gram-negative
bacteria by LPS (13, 25) together with CD14 (26). Recently, another molecule, MD2, has been shown to interact with the ectodomain of TLR4 to confer LPS responsiveness, an essential prerequisite for TLR4 signaling (27). Engagement of TLRs by
microbial products results in homodimerization and recruitment of
the adaptor molecule MyD88 (28), the functional homolog of Drosophila adaptor molecule Tube. In mammals, engagement of
MyD88 leads to NF-␬B nuclear translocation (19, 29).
Porins are of particular interest because they have been characterized as potent adjuvants and have great potential as a novel
component of vaccines. Neisserial porins belong to the Gram-negative porin superfamily (30) and share significant structural similarities with other members of the family, resembling other
P.H. is supported in part by Deutsche Forschungsgemeinschaft (He 3127/1-1).
2
Address correspondence and reprint requests to Dr. Lee M. Wetzler, Department of
Medicine, Division of Infectious Diseases, Boston University School of Medicine,
650 Albany Street, Boston, MA 02118. E-mail address: [email protected]
Copyright © 2002 by The American Association of Immunologists
●
3
Abbreviations used in this paper: TLR, Toll-like receptor; DC, dendritic cell;
PAMP, pathogen-associated molecular pattern; CHO, Chinese hamster ovary; HEK,
human embryonal kidney.
0022-1767/02/$02.00
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The immunopotentiating activity of neisserial porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the
surface expression of B7-2. This ability is dependent on MyD88
and Toll-like receptor (TLR)2 expression, as demonstrated by
a lack of a response by B cells from MyD88 or TLR2 knockout
mice to the porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and
were shown to be due to induction of NF-␬B nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2. The Journal of Immunology, 2002, 168: 1533–1537.
1534
CUTTING EDGE: MENINGOCOCCAL PORIN PorB IS A TLR2 LIGAND
Materials and Methods
Animal strains
C57BL/6 wild-type mice and TLR2 knockout mice (34) or C57BL/6 and
MyD88 knockout mice (35) were used.
Lymphocyte isolation, cell lines, and constructs
B lymphocytes were purified from splenocytes as described (10). A Chinese hamster ovary (CHO-K1) reporter cell line, clone 3E10 (36), stably
transfected with human CD14 and an NF-␬B-driven reporter construct,
which regulates the surface expression of Tac (CD25) Ag (22). Derivatives
of 3E10 were used: clone 7.19 (27) and clone 7.19-TLR2, which expresses
human TLR2 (P. Henneke, unpublished results) (23, 27, 36). Human embryonal kidney (HEK) cells (HEK-293) were transiently transfected with a
pELAM-luciferase NF-␬B reporter construct (13) plus either human TLR2
or the empty vector using the Polyfect transfection reagent (Qiagen,
Valencia, CA).
Results
Neisserial porin stimulation of B cells is MyD88 dependent
Purified splenic B cells from C57BL/6 wild-type and MyD88
knockout mice were incubated with purified meningococcal PorB
or with medium alone. After 24 h, the levels of surface expression
of B7-2 and class II MHC were measured by flow cytometry (Fig.
1A). PorB failed to mediate up-regulation of B7-2 or class II MHC
on B cells from MyD88 knockout mice. B cells from C57BL/6
wild-type mice are responsive to LPS, due to the expression of
intact TLR4 on their surface. In previous studies by our group (10)
we have found that neisserial porin preparations do not appear to
contain a significant amount of LPS, which may account for the
induction of B7-2 surface expression. However, B cells from
C57BL/6 wild-type mice and MyD88 knockout mice were also
incubated with PorB in the presence of E5564, a TLR4 antagonist
(39). E5564 abrogated the effect of LPS on B cells from C57BL/6
wild-type mice but did not modify the effect of neisserial porins, as
shown in Fig. 1, A and B. Controls for LPS-mediated activation of
B cells were performed by incubating the cells with N. meningitidis LPS, which could not induce expression of B7-2 or class II
MHC in MyD88-deficient B cells (Fig. 2A). Similar results were
obtained using purified Escherichia coli LPS (data not shown).
These data demonstrate that B cell activation by neisserial porins
is MyD88 dependent.
Dendritic cells (DCs) from MyD88 knockout mice have been
shown to respond to LPS with delayed kinetics (40); however, no
data are available on the behavior of purified splenic B cells from
MyD88 knockout mice in response to LPS or other bacterial products. Therefore, longer incubations with neisserial porins were performed to determine whether a similar MyD88-independent stimulation pathway exists for neisserial porins. Purified B cells were
incubated for 48 h with PorB or meningococcal LPS in the presence or absence of E5564, and the up-regulation of expression of
B7-2 and class II MHC was measured by flow cytometric analysis.
The histograms in Fig. 1B show that PorB was not able to induce
increased surface expression of B7-2 or class II MHC in MyD88deficient B cells after 48 h of incubation. Incubation with LPS for
Neisserial porins and reagents
PorB was purified from N. meningitidis strain H44/76 lacking both PorA
and RmpM (10, 37) as described (1). Purified neisserial LPS (kindly provided by Dr. M. Apicella, University of Iowa Medical Center, Iowa City,
IA) was depleted of contaminating lipoproteins by phenol extraction
method (38). The LPS inhibitor, compound E5564, was provided by Eisai
Research Institute (Andover, MA; patent reference no. WO-9639411-A1).
The following anti-murine mAbs were used for flow cytometric analysis:
anti-rat IgG, anti-CD86 (B7-2), anti-class II MHC (IAb), and anti-CD25,
all FITC conjugated (BD PharMingen, San Diego, CA).
Cell incubations and flow cytometric analysis
B lymphocytes (5 ⫻ 106/ml) were incubated with 10 ␮g/ml PorB or 100
ng/ml N. meningitidis LPS in the presence or absence of 10 ␮g/ml E5564.
CHO reporter cell lines (105/ml) were incubated with 20 ␮g/ml PorB or
with 5 ng/ml IL-1␤. The expression of surface Ags was examined by flow
cytometric analysis (10, 36).
NF-␬B luciferase reporter assay
HEK/TLR2 cells (5 ⫻ 105/well) were incubated for 16 h with 0.1, 1, and
10 ␮g/ml PorB, heat-killed Listeria monocytogenes, or 5 ng/ml IL-1␤. Cell
activation was determined by measuring luciferase activity of the total
cellular lysate using an assay kit from Promega (Madison, WI) according
to the manufacturer’s instructions. The data are reported as the mean of
triplicate determinations ⫾ SD.
FIGURE 1. PorB does not stimulate MyD88-deficient B cells. C57BL/6
wild-type and MyD88-deficient B cells were incubated for 24 (A) or 48 h
(B) with medium (shaded histograms), 10 ␮g/ml PorB alone (solid lines),
or 10 ␮g/ml E5564 (gray lines). The surface expression of B7-2 and class
II MHC was determined by flow cytometric analysis.
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PAMPs. Given the importance of mammalian TLRs in innate immunity, including their role in B7 up-regulation, and in the light of
their ability to discriminate between different pathogens and bacterial
products, their involvement in the response to neisserial porins was
investigated. Thus far, the involvement of TLRs on the effect of immune adjuvants has only been theorized (16). However, one known
immune adjuvant, unmethylated bacterial CpG DNA motifs, appears
to induce its immunopotentiating effect through TLR9 (31).
Previous experimental evidence from studies using C3H/HeJ
mice established that the immune response to neisserial porins is
not abrogated in the absence of functional TLR4. Thus, the involvement of other TLRs in the cellular response to neisserial porins was investigated. Recent reports regarding the participation of
TLR2 in response to Neisseria meningitidis (32, 33) suggested that
outer membrane components of meningococcus can activate immune cells by engaging TLR2. However, the specific role of porins
was not determined. B cells from TLR2 knockout mice (34) and
MyD88 knockout mice (35) were used to elucidate the involvement of these molecules in the ability of the porin to stimulate B
cells and induce increased surface expression of B7-2 and class II
MHC. The data presented in this study directly demonstrate the
importance of TLR2 in the adaptive immune response induced by
potential vaccine adjuvants.
The Journal of Immunology
48 h also did not induce increased surface expression of B7-2 or
class II MHC in MyD88-deficient B cells (Fig. 2B).
Neisserial porins induce TLR2-mediated NF-␬B nuclear
translocation
To investigate the interaction of neisserial porins with TLR2, two
different cellular reporter constructs were used to measure the
TLR2-mediated NF-␬B nuclear translocation induced by neisserial
porins. CHO cells, normally unresponsive to LPS, were transfected
with CD14 (36) and a reporter gene encoding CD25 (Tac) (22).
Among such transfectants, clone 7.19 (27), which had a negligible
response to LPS (36), was transfected with a plasmid containing
human tlr2 gene, and 7.19/TLR2 cells were incubated for 16 h
with PorB or IL-1␤ as a TLR2-independent control. Fig. 3A shows
FIGURE 3. PorB induces TLR2-mediated NF-␬B translocation. A,
CHO cells, clones 7.19 and 7.19/TLR2, incubated with PBS, 20 ␮g/ml
PorB, or 5 ng/ml IL-1␤ for 16 h, were analyzed by flow cytometry for
CD25 expression. B, HEK/TLR2 cells were incubated for 5 h with IL-1␤
(5 ng/ml), heat-killed L. monocytogenes, or PorB (0.1–10 ␮g/ml). NF-␬B
translocation was determined by luciferase production. HEK cells transfected with a naked vector were used as a negative control.
the level of CD25 surface expression, determined by flow cytometric analysis using anti-CD25 FITC-labeled Ab. PorB induced
increased expression of CD25 on the surface of TLR2 transfected
cells, while it failed to induce CD25 expression in the cells that
were not transfected with TLR2.
HEK-293 cells, which do not normally express TLR2, TLR4, or
MD-2 (41), were transiently cotransfected with an NF-␬B-dependent luciferase reporter plasmid (13) and with the human tlr2 gene.
Cells were incubated with different concentrations of PorB and, as
a TLR2-dependent positive control, with heat-killed L. monocytogenes. IL-1␤ incubation was also used as a control, and the naked
vectors were used as a negative control. Cell lysates were obtained
and incubated in the presence of luciferin as described. As shown
in Fig. 3B, PorB induced a dose-dependent increase in luminescence only in cells transfected with TLR2. Increasing concentrations of meningococcal LPS (up to 1 ␮g/ml) failed to stimulate
those cells, while TLR4/MD-2-expressing cells were stimulated by
LPS but were not stimulated by PorB (data not shown). These
latter data confirm that the presence of LPS in the porin proteosomes preparations was minimal and its effect in these experiments
was negligible.
TLR2 is essential for neisserial porin up-regulation of B7-2 and
class II MHC in B cells
B cells were isolated from spleens of TLR2 knockout mice (34) in
a C57BL/6 background and from C57BL/6 wild-type mice, and
were incubated with PorB or medium alone. After 24 h, the level
of surface expression of B7-2 and class II MHC was determined by
flow cytometric analysis. As shown in Fig. 4A, PorB failed to
up-regulate the surface expression of B7-2 or class II MHC on B
cells from TLR2 knockout mice, as compared with B cells from
wild-type mice, already shown in Figs. 1 and 2. This indicates that
the up-regulation of B7-2 and class II MHC surface expression
was dependent on the normal expression of TLR2 on the B cell.
B cells from TLR2 knockout mice were also incubated with
PorB in the presence of E5564, which did not affect the expression
of B7-2 or class II MHC in porin-treated cells (Fig. 4A), indicating
that B cell activation was not mediated by engagement of TLR4.
These data have been confirmed in another LPS-sensitive mouse
strain, C3H/OuJ, in which the up-regulation of B7-2 and class II
MHC induced by neisserial porins was not affected by the presence
of this LPS inhibitor (data not shown).
As a control for the involvement of TLR4-mediated LPS stimulation, B cells from TLR2 knockout mice were incubated with
FIGURE 4. PorB does not stimulate TLR2-deficient B cells as compared to LPS. A, B cells from TLR2 knockout mice were incubated for 24 h
with medium (shaded histogram) or 10 ␮g/ml N. meningitidis PorB alone
(solid line) or 10 ␮g/ml E5564 (gray line). B, B cells were incubated as
above, with medium or 100 ng/ml N. meningitidis LPS alone (solid line) or
E5564 (gray line). C57BL/6 control B cells are shown in Figs. 1 and 2. The
surface expression of B7-2 and class II MHC (IAb)was determined by flow
cytometric analysis.
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FIGURE 2. Effect of LPS on MyD88-deficient B cells. C57BL/6 wildtype and MyD88-deficient B cells were incubated for 24 (A) or 48 h (B)
with medium (shaded histograms), 100 ng/ml N. meningitidis LPS alone
(solid line), or 10 ␮g/ml E5564 (gray lines), and the surface expression of
B7-2 and class II MHC was determined by flow cytometric analysis.
1535
1536
CUTTING EDGE: MENINGOCOCCAL PORIN PorB IS A TLR2 LIGAND
Discussion
Neisserial porins are known to act as immune adjuvants by upregulating the surface expression of the costimulatory molecule
B7-2 on B cells and probably other APCs (4, 10). Until now, there
has been no clear evidence regarding the modality of interaction of
purified neisserial porins with eukaryotic cells, and no receptorligand model has been characterized. Indeed, the physical characteristics of porin, as a pore-forming molecule in bacterial membranes, suggested that porins might activate immune cells by
forming ion channels in mammalian membranes. The purpose of
this study was to examine the possibility that Toll-like receptors
function as cognate receptors for neisserial porins on B cells.
MyD88 is an effector molecule associated with TLR-mediated
response in lymphoid cells. The use of MyD88 knockout mice has
shown that the activation of B cells by neisserial porin is strictly
MyD88 dependent. It has been demonstrated that MyD88-deficient
DCs are still able to respond to LPS via TLR4, but with delayed
kinetics (40), due to the presence of a new TLR adaptor protein,
termed TLR/IL-1R-associated protein (42). To investigate the possibility of a MyD88-independent pathway for neisserial porin signaling, we used meningococcal PorB or LPS for extended time
points. However, we could not detect up-regulation of B7-2 or
class II MHC even after 48 h of incubation, suggesting that their
signaling is strictly dependent on MyD88. These data suggested
involvement of TLR in the immune stimulation of neisserial
porins.
The role of TLRs, specifically TLR2 and TLR4, in the innate
and adaptive immune responses has been examined in detail by
several investigators (43, 44). In the past few years, the activation
of B cells and DCs in response to different pathogens and bacterial
products has been correlated to the presence of different TLRs on
the cell surface. Specifically, TLR2 and TLR4 can recognize a
series of ligands, such as bacterial lipopeptides, LPS, and other
outer membrane bacterial components (17, 19, 23, 24), thus initiating signaling pathways which affect the immune response, such
as up-regulation of B7 molecules on the surface of B cells and DCs
or nuclear translocation of the transcription factor NF-␬B. The
evidence that TLR4 is not critical for the signaling pathway initiated by neisserial porins preceded many of the fundamental discoveries of the TLRs. Neisserial porins induce increased surface
expression of B7-2 on B cells from C3H/HeJ mice, which are
unresponsive to LPS because of a TLR4 mutation (12), as well as
in their LPS-sensitive counterpart, C3H/OuJ mice (10, 11). The
possibility that TLR2 might be involved in response to neisserial
porins was investigated, because this TLR appears to have an extraordinarily broad repertoire of ligands. All the lines of evidence
suggested a central role for TLR2 in porin recognition.
TLR2 is known to be a receptor for bacterial lipoproteins and
their interaction has been shown to induce B7-2 surface expression
on B cells (16). Outer membrane preparations may contain variable amounts of lipoproteins. Biochemical analysis and in vitro
studies comparing neisserial porin preparations with purified neisserial lipopeptide indicated that the effect of porin was not related
to the presence of lipopeptide, supporting our hypothesis that the
TLR2-mediated up-regulation of B7-2 and class II MHC on B cells
surface by neisserial porins mediates their adjuvant activity on the
immune response.
In conclusion, this work demonstrates that the effect of neisserial porins on the surface expression of B7-2 and class II MHC on
B cells, which mediates the effect of these proteins, is dependent
on the interaction of porins with TLR2 on the cell surface. Also,
the presence of the effector molecule MyD88 is required for the
activity of the porin. Furthermore, we have shown that the effect of
neisserial porin on B cells is dependent on MyD88, and that a
delayed response to neisserial porins or LPS was not detectable in
B cells, as compared with previous reports regarding the effect of
LPS on MyD88-deficient DCs (40). As we have demonstrated that
the adjuvant activity of neisserial porins is specifically related to
their ability to induce increased B7-2 surface expression on APCs,
this work suggests that their adjuvant effect is, therefore, dependent on porin-mediated TLR2 signaling events.
Acknowledgments
We thank Drs. Sanjay Ram and Robin R. Ingalls for helpful discussion. We
also thank Dr. S. Akira (Osaka University, Osaka, Japan) for the use of
TLR2 and MyD88 knockout mice.
References
1. Wetzler, L. M., M. S. Blake, K. Barry, and E. C. Gotschlich. 1992. Gonococcal
porin vaccine evaluation: comparison of Por proteosomes, liposomes and blebs
isolated from rmp deletion mutants. J. Infect. Dis. 166:551.
2. Wetzler, L. M. 1994. Immunopotentiating ability of neisserial major outer membrane proteins: use as an adjuvant for poorly immunogenic substances and potential use in vaccines. Ann. NY Acad. Sci. 730:367.
3. Lowell, G. H., W. R. Ballou, L. F. Smith, R. A. Wirtz, W. D. Zollinger, and
W. T. Hockmeyer. 1988. Proteosome-lipopeptide vaccines: enhancement of immunogenicity for malaria CS peptides. Science 240:800.
4. Mackinnon, F. G., Y. Ho, M. S. Blake, F. Michon, A. Chandraker, M. H. Sayegh,
and L. M. Wetzler. 1999. The role of B/T costimulatory signals in the immunopotentiating activity of neisserial porin. J. Infect. Dis. 180:755.
5. Donnelly, J. J., R. R. Deck, and M. A. Liu. 1990. Immunogenicity of a Haemophilus influenzae polysaccharide-Neisseria meningitidis outer membrane protein vaccine. J. Immunol. 145:3071.
6. Giebink, G. S., M. Koskela, P. P. Vella, M. Harris, and C. T. Le. 1993. Pneumococcal capsular polysaccharide-meningococcal outer membrane protein complex conjugate vaccines: immunogenicity and efficacy in experimental pneumococcal otitis media. J. Infect. Dis. 167:347.
7. Fusco, P. C., F. Michon, J. Y. Tai, and M. S. Blake. 1997. Preclinical evaluation
of a novel group B meningococcal conjugate vaccine that elicits bactericidal
activity in both mice and nonhuman primates. J. Infect. Dis. 175:364.
8. Livingston, P. O., M. J. Calves, F. Helling, W. D. Zollinger, M. S. Blake, and
G. H. Lowell. 1993. GD3/proteosome vaccines induce consistent IgM antibodies
against the ganglioside GD3. Vaccine 11:1199.
9. Lowell, G. H., L. F. Smith, R. C. Seid, and W. D. Zollinger. 1988. Peptides bound
to proteosomes via hydrophobic feet become highly immunogenic without adjuvant. J. Exp. Med. 167:658.
10. Wetzler, L. M., Y. Ho, and H. Reiser. 1996. Neisserial porins induce B lymphocytes to express costimulatory B7-2 molecules and to proliferate. J. Exp. Med.
183:1151.
11. Bhasin, N., Y. Ho, and L. M. Wetzler. 2001. Neisseria meningitidis lipopolysaccharide modulates the specific humoral immune response to neisserial porins but
has no effect on porin-induced upregulation of costimulatory ligand B7-2. Infect.
Immun. 69:5031.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
purified LPS from N. meningitidis in the presence or absence of
E5564. Up-regulation of B7-2 expression and class II MHC on the
B cell surface of TLR2 knockout mice induced by incubation with
LPS was inhibited by the cotreatment with E5564 (Fig. 4B). Virtually identical results were obtained when E. coli LPS was used
to stimulate the B cells (data not shown). These data indicate that
the TLR4 pathway was not involved in the effect of neisserial
porins on B7-2 or class II MHC expression in B cells but is solely
involved in the effect of LPS on the expression of these ligands.
The increased surface expression of B7-2 and class II MHC on
B cells from TLR2 knockout mice or C57BL/6 wild-type mice
induced by purified neisserial lipopeptide (a gift from P. Fisette
and Dr. R. Ingalls, Boston University School of Medicine, Boston,
MA), a common TLR2 ligand, was compared with the effect of
neisserial porin preparations. Using high doses of purified lipopeptide, stimulation of B cells was achieved. Low doses of purified
lipopeptide (comparable to the lipopeptide content of neisserial
porin preparations, as demonstrated by immunoblot analysis)
failed to induce increased expression of B7-2 and class II MHC on
the B cell surface (data not shown), suggesting that the up-regulation of these molecules was due to the immune modulatory properties of neisserial porin and not due to potential lipoprotein
contamination.
The Journal of Immunology
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point
mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.
J. Exp. Med. 194:79.
Kawai, T., O. Adachi, T. Ogawa, K. Takeda, and S. Akira. 1999. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115.
Medzhitov, R., and C. A. Janeway, Jr. 1999. Innate immune induction of the
adaptive immune response. Cold Spring Harbor Symp. Quant. Biol. 64:429.
Jeanteur, D., J. H. Lakey, and F. Pattus. 1991. The bacterial porin superfamily:
sequence alignment and structure prediction. Mol. Microbiol. 5:2153.
Krieg, A. M., and H. L. Davis. 2001. Enhancing vaccines with immune stimulatory CpG DNA. Curr. Opin. Mol. Ther. 3:15.
Pridmore, A. C., D. H. Wyllie, F. Abdillahi, L. Steeghs, P. van der Ley,
S. K. Dower, and R. C. Read. 2001. A lipopolysaccharide-deficient mutant of
Neisseria meningitidis elicits attenuated cytokine release by human macrophages
and signals via Toll-like receptor (TLR)2 but not via TLR4/MD2. J. Infect. Dis.
183:89.
Ingalls, R. R., E. Lien, and D. T. Golenbock. 2001. Membrane-associated proteins of a lipopolysaccharide-deficient mutant of Neisseria meningitidis activate
the inflammatory response through Toll-like receptor 2. Infect. Immun. 69:2230.
Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda,
and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of gramnegative and gram-positive bacterial cell wall components. Immunity 11:443.
Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami,
K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results
in loss of IL-1- and IL-18-mediated function. Immunity 9:143.
Delude, R. L., A. Yoshimura, R. R. Ingalls, and D. T. Golenbock. 1998. Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-␣, signal transduction. J. Immunol.
161:3001.
Guttormsen, H. K., L. M. Wetzler, and A. Naess. 1993. Humoral immune response to the class 3 outer membrane protein during the course of meningococcal
disease. Infect. Immun. 61:4734.
Hirschfeld, M., Y. Ma, J. H. Weis, S. N. Vogel, and J. J. Weis. 2000. Cutting
edge: repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. J. Immunol. 165:618.
Ingalls, R. R., B. G. Monks, R. J. Savedra, W. J. Christ, R. L. Delude,
A. E. Medvedev, T. Espevik, and D. T. Golenbock. 1998. CD11/CD18 and CD14
share a common lipid A signaling pathway. J. Immunol. 161:5413.
Kaisho, T., O. Takeuchi, T. Kawai, K. Hoshino, and S. Akira. 2001. Endotoxininduced maturation of MyD88-deficient dendritic cells. J. Immunol. 166:5688.
da Silva Correia, J., K. Soldau, U. Christen, P. S. Tobias, and R. J. Ulevitch.
2001. Lipopolysaccharide is in close proximity to each of the proteins in its
membrane receptor complex: transfer from CD14 to TLR4 and MD-2. J. Biol.
Chem. 276:21129.
Horng, T., G. M. Barton, and R. Medzhitov. 2001. TIRAP: an adapter molecule
in the Toll signaling pathway. Nat. Immunol. 2:835.
Takeuchi, O., and S. Akira. 2001. Toll-like receptors: their physiological role and
signal transduction system. Int. Immunopharmacol. 1:625.
Ozinsky, A., D. M. Underhill, J. D. Fontenot, A. M. Hajjar, K. D. Smith,
C. B. Wilson, L. Schroeder, and A. Aderem. 2000. The repertoire for pattern
recognition of pathogens by the innate immune system is defined by cooperation
between Toll-like receptors. Proc. Natl. Acad. Sci. USA 97:13766.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
12. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. V. Huffel, X. Du, D. Birdwell,
E. Alejos, M. Silva, C. Galanos, et al. 1998. Defective LPS signaling in C3H/HeJ
and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085.
13. Chow, J. C., D. W. Young, D. T. Golenbock, W. J. Christ, and F. Gusovsky.
1999. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274:10689.
14. Hashimoto, C., K. L. Hudson, and K. V. Anderson. 1988. The Toll gene of
Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a
transmembrane protein. Cell 52:269.
15. Lemaitre, B., E. Nicolas, L. Michaut, J. M. Reichhart, and J. A. Hoffmann. 1996.
The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent
antifungal response in Drosophila adults. Cell 86:973.
16. Medzhitov, R., P. Preston-Hurlburt, and C. A. Janeway, Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.
Nature 388:394.
17. Faure, E., L. Thomas, H. Xu, A. Medvedev, O. Equils, and M. Arditi. 2001.
Bacterial lipopolysaccharide and IFN-␥ induce Toll-like receptor 2 and Toll-like
receptor 4 expression in human endothelial cells: role of NF-␬B activation. J. Immunol. 166:2018.
18. Medzhitov, R., and C. A. J. Janeway. 1997. Innate immunity: the virtues of a
nonclonal system of recognition. Cell 91:295.
19. Underhill, D. M., A. Ozinsky, A. M. Hajjar, A. Stevens, C. B. Wilson,
M. Bassetti, and A. Aderem. 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401:811.
20. Poltorak, A., I. Smirnova, X. He, M. Y. Liu, C. Van Huffel, O. McNally,
D. Birdwell, E. Alejos, M. Silva, X. Du, et al. 1998. Genetic and physical mapping of the Lps locus: identification of the Toll-4 receptor as a candidate gene in
the critical region. [Published erratum appears in 1999 Blood Cells Mol. Dis.
25:78.] Blood Cells Mol. Dis. 24:340.
21. Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, and
D. Golenbock. 1999. Cutting edge: recognition of Gram-positive bacterial cell
wall components by the innate immune system occurs via Toll-like receptor 2.
J. Immunol. 163:1.
22. Flo, T. H., O. Halaas, E. Lien, L. Ryan, G. Teti, D. T. Golenbock, A. Sundan, and
T. Espevik. 2000. Human Toll-like receptor 2 mediates monocyte activation by
Listeria monocytogenes, but not by group B streptococci or lipopolysaccharide.
J. Immunol. 164:2064.
23. Lien, E., T. J. Sellati, A. Yoshimura, T. H. Flo, G. Rawadi, R. W. Finberg,
J. D. Carroll, T. Espevik, R. R. Ingalls, J. D. Radolf, and D. T. Golenbock. 1999.
Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J. Biol. Chem. 274:33419.
24. Brightbill, H. D., D. H. Libraty, S. R. Krutzik, R. B. Yang, J. T. Belisle,
J. R. Bleharski, M. Maitland, M. V. Norgard, S. E. Plevy, S. T. Smale, et al. 1999.
Host defense mechanisms triggered by microbial lipoproteins through Toll-like
receptors. Science 285:732.
25. Heine, H., C. J. Kirschning, E. Lien, B. G. Monks, M. Rothe, and
D. T. Golenbock. 1999. Cutting edge: cells that carry a null allele for Toll-like
receptor 2 are capable of responding to endotoxin. J. Immunol. 162:6971.
26. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, and J. C. Mathison.
1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS
binding protein. Science 249:1431.
27. Schromm, A. B., E. Lien, P. Henneke, J. C. Chow, A. Yoshimura, H. Heine,
E. Latz, B. G. Monks, D. A. Schwartz, K. Miyake, and D. T. Golenbock. 2001.
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