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IMMUNOBIOLOGY
From the Department of Immunology, Saga Medical School,
Japan; the Center for Experimental Medicine, Division of Infectious
Genetics, The Institute of Medical Science, The University of Tokyo,
Japan; and the Department of Immunology, Faculty of Medicine, Tottori
University, Yonago, Japan.
RP105 is a B-cell surface molecule that has been recently assigned
as CD180. RP105 ligation with an antibody induces B-cell activation in
humans and mice, leading to proliferation and up-regulation of a
costimulatory molecule, B7.2/CD86. RP105 is associated with an
extracellular molecule, MD-1. RP105/MD-1 has structural similarity to
Toll-like receptor 4 (TLR4)/MD-2. TLR4 signals a membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS). MD-2 is
indispensable for TLR4-dependent LPS responses because cells expressing
TLR4/MD-2, but not TLR4 alone, respond to LPS. RP105 also has a role in
LPS responses because B cells lacking RP105 show hyporesponsiveness to
LPS. Little is known, however, regarding whether MD-1 is important for
RP105-dependent LPS responses, as MD-2 is for TLR4. To address the
issue, we developed mice lacking MD-1 and generated monoclonal antibodies (mAbs) to the protein. MD-1-null mice showed impairment in
LPS-induced B-cell proliferation, antibody production, and B7.2/CD86
up-regulation. These phenotypes are similar to those of RP105-null
mice. The similarity was attributed to the absence of cell surface
RP105 on MD-1-null B cells. MD-1 is indispensable for cell surface
expression of RP105. A role for MD-1 in LPS responses was further
studied with anti-mouse MD-1 mAbs. In contrast to highly mitogenic
anti-RP105 mAbs, the mAbs to MD-1 were not mitogenic but antagonistic
on LPS-induced B-cell proliferation and on B7.2 up-regulation.
Collectively, MD-1 is important for RP105 with respect to B-cell
surface expression and LPS recognition and signaling.
(Blood. 2002;99:1699-1705) The innate immune response is the first line of
defense against microbial pathogens.1-3 The principal
challenge for the immune system is to recognize pathogens and to mount
an immediate defense response. A wide variety of bacterial components
are capable of stimulating the innate immune system. These include
lipopolysaccharide (LPS), peptidoglycan, lipoteichoic acid,
lipoarabinomannan, lipopeptides, and bacterial DNA. LPS is a principal
component of Gram-negative bacteria that potently activates the innate
immune system, and it is one of the best-studied molecules among
microbial products.4
Toll-like receptors (TLRs), mammalian homologues of
Drosophila Toll, have been implicated in innate
recognition and signaling of these microbial products in humans and
mice.5,6 TLR4, for example, signals LPS. The mutations of
the TLR4 gene lead to LPS hyporesponsiveness in humans and
mice.7-10 TLR4 expression, however, does not confer LPS
responsiveness on cell lines, suggesting a requirement for an
additional molecule.11 We recently cloned a molecule,
MD-2, that is associated with the extracellular domain of TLR4.
Coexpression of MD-2 imparts LPS responsiveness to TLR4.11
MD-2 was originally identified as a molecule similar to MD-1. MD-1 is
associated with the extracellular domain of RP105.12 RP105
is a type 1 transmembrane protein with extracellular leucine-rich repeats (LRRs) and a short cytoplasmic tail.13 RP105 is
similar to Drosophila Toll in the extracellular LRRs. In
this regard, RP105 is the first mammalian molecule to be described as
similar to Toll. In contrast to ubiquitous expression of TLR4, RP105
expression is largely restricted to immune cells including mature B
cells and macrophages.13 Antibody-mediated cross-linking
of RP105 induces resistance against irradiation-induced apoptosis,
robust B-cell proliferation, and up-regulation of a costimulatory
molecule, B7.2, revealing RP105 as a potent regulator of B-cell
activation.14-17 RP105 has an important role in B-cell
activation by LPS because B cells lacking RP105 are impaired in
LPS-induced proliferation and antibody production.18
Little is known, however, about a role for MD-1 in RP105-dependent LPS
responses. We addressed the issue by establishing monoclonal antibodies
(mAbs) to mouse MD-1 and mice lacking MD-1.
Animals
Cells and derivation of the monoclonal antibodies to mouse
MD-1
Rats were immunized with NRK cells expressing mouse RP105/MD-1, and
mAbs were screened that stained RP105/MD-1-expressing Ba/F3 cells
specifically. We next excluded the mAbs reactive with RP105 by
selecting the mAbs that stained Ba/F3 cells expressing human
RP105/mouse MD-1, but not those expressing human RP105/human MD-1
(Figure 1).9 Monoclonal antibodies were cloned and
purified for further studies. We used 2 mAbs, MD14 (rat IgG2a/ Generation of the MD-1-deficient mice
Cell staining and flow cytometry
Analysis of in vitro B-cell activation Splenic B cells were purified by negative selection with the anti-CD43 mAb S7 conjugated to Dynabeads M-450 sheep anti-rat IgG (Dynal, Lake Success, NY) as described.18 B-cell purity has been always higher than 95%, as judged by flow cytometry analyses (data not shown). Wild-type and mutant B cells were incubated at 2 × 105/well in 96-well, flat-bottomed plates with: goat anti-mouse IgM F(ab')2 (Organon Teknika, Tokyo, Japan); rat anti-mouse CD40 mAb LB429S23; rat anti-mouse RP105 mAb RP/14; or LPS. For the analysis of LPS responses, B cells were treated with LPS derived from Salmonella minnesota Re595 or Escherichia coli 055:B5. Lipid A was of bacterial origin (S minnesota). For proliferation, B cells were stimulated for 3 days, pulsed with 1 µCi/well (37 000 Bq) [3H]-TdR, and harvested onto a glass filter. The uptake of [3H]-TdR was counted as described previously.18Analysis of humoral immune responses Littermate control and MD-1-deficient mice were immunized intraperitoneally with 20 µg trinitrophenyl (TNP)-LPS in phosphate-buffered saline (5 mice per group). Serum concentrations of TNP-specific antibodies at different time points were measured by enzyme-linked immunosorbent assay (ELISA). ELISA was performed by coating plastic plates with TNP-bovine serum albumin (10 µg/mL), and serial serum dilutions were applied onto the plate. Bound antibodies were revealed by goat antibodies specific for IgM or IgG3 isotype (Caltag Laboratories, Burlingame, CA).Statistical analysis Significance was evaluated using the Student t test for unpaired data.Immunoprecipitation and immunoprobing The interleukin-3 (IL-3)-dependent line Ba/F3 was transfected with the pEFBOS expression vector encoding a variety of mouse TLR4, mouse MD-2, mouse RP105, and mouse MD-1. Ba/F3 cells expressing RP105/MD-1; RP105 and MD-2; and RP105/MD-1 + TLR4/MD-2 were established as described previously.11,18 Cells were washed and lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 50 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL soybean trypsin inhibitor, 2 mM MgCl2, and 2 mM CaCl2. After 30 minutes of incubation on ice, the lysate was centrifuged and nuclei were removed. Beads coupled with anti-RP105 mAb RP/14 or with the anti-flag mAb M2 were added to the cell lysate and were rotated for 4 hours at 4°C. Beads were washed in the lysis buffer, and bound proteins were subjected to SDS-PAGE (10% acrylamide under nonreduced conditions) and Western blot analysis. Precipitated proteins were detected with the anti-flag mAb M2 (Sigma, St Louis, MO) and Supersignal chemiluminescent substrate (Pierce, Rockford IL). Cell surface biotinylation was also used to detect RP105, as described previously.13
Derivation of monoclonal antibodies to mouse MD-1 To detect cell surface MD-1, we made mAbs to mouse MD-1 and showed the specificity of mAbs by fluorescence staining (Figure 1). Anti-mouse MD-1 mAbs reacted with mouse RP105/MD-1 but did not cross-react with human RP105/MD-1. Whereas the anti-RP105 mAb did not react with the complex consisting of human RP105 and mouse MD-1, the anti-MD-1 mAbs did (Figure 1B,E), confirming their specificity to MD-1. Anti-MD-1 mAbs were used for fluorescence staining of spleen cells. Positive cells were mainly restricted to mature B cells (Figure 2C and data not shown), which were similar to the results with the anti-RP105 mAb.14B cells lacking MD-1 are hyporesponsive to lipopolysaccharide To address a role of MD-1 in the immune system, we made mice lacking MD-1. The first exon encodes a signal sequence and the following 27 amino acids. The exon was replaced with the neomycin resistance gene in the targeted allele (Figure 2A). Truncated products, even if aberrantly produced, should stay inside the cells. Mice homozygous with the targeted allele, which was confirmed with Southern hybridization (Figure 2B), did not express MD-1 as judged by flow cytometry staining of spleen cells with the anti-MD-1 mAb (Figure 2C).Flow cytometry analyses of lymphoid organs, including the bone marrow, spleen, lymph node, and thymus, did not reveal any differences between homozygous and wild-type mice, demonstrating normal lymphocyte and myeloid cell development in the absence of MD-1 (data not shown). Because MD-1 was expressed on mature spleen B cells, we stained a variety of B cell markers, including CD45, CD21, CD22, CD23, surface IgM, and surface IgD. Again, we could not see any difference between wild-type and MD-1-null B cells. Splenic B cell maturation was not altered in the absence of MD-1. B-cell responsiveness to a variety of stimuli was next studied.
MD-1-deficient B cells were not impaired in proliferation induced by
anti-CD40 or anti-IgM antibody, whereas they revealed low responses in
proliferation driven by anti-RP105 mAb, lipid A, or LPS prepared from
S minnesota or E coli (Figure
3). These results prompted us to further
study B-cell responses to LPS or the anti-RP105 mAb. In addition to
proliferation, LPS-induced B-cell activation led to up-regulation of a
variety of cell surface molecules, such as B7.2/CD86. B7.2 is a
costimulatory molecule that facilitates an interaction with T cells
(reviewed in Lenschow et al24). LPS, therefore, acts as an
adjuvant in this context. Lipid A or anti-RP105 mAb induced B7.2
up-regulation on wild-type B cells, but not on those lacking MD-1
(Figure 4). The lack of MD-1, however,
did not affect anti-IgM-induced B7.2 up-regulation, revealing the
selective impairment in the responses to LPS or the anti-RP105 mAb
(Figure 4). Not all LPS responses were impaired in B cells lacking
MD-1. CD23 is a B-cell activation antigen involved in the regulation of
B-cell proliferation and the production of IgE. Interestingly, CD23 was
up-regulated with lipid A but not with the anti-RP105 mAb or anti-IgM
Ab. Lipid A-induced up-regulation of CD23 was not impaired in
MD-1-null mice (Figure 4, and "Discussion").
We also studied an in vivo B-cell response to LPS. Mice were immunized
with TNP-LPS, and the production of TNP-specific antibodies was
measured with ELISA. We show the results of IgM or IgG3 isotypes, which
were dominant in response to TNP-LPS. Consistent with in vitro studies,
MD-1-deficient mice revealed hyporesponsiveness in antibody production
to TNP-LPS (Figure 5). We also studied antibody responses to a thymus-independent type 2 antigen TNP-Ficoll. MD-1-deficient mice were not affected in the antibody response (data
not shown).
MD-1 is indispensable for RP105 expression on the B-cell surface Our results clearly showed that B cells lacking MD-1 were hyporesponsive to LPS or anti-RP105 mAb. In this regard, B cells lacking MD-1 are similar to RP105-null B cells.18 RP105 and MD-1 are associated with each other, and we previously showed that transfected MD-1 up-regulated cell surface RP105 that had been transfected beforehand,12,15 suggesting an important role for MD-1 in cell surface expression of RP105. LPS hyporesponsiveness in B cells lacking MD-1 may be attributed to impaired expression of RP105. We stained RP105 on B cells lacking MD-1 and found the complete absence of RP105 (Figure 6). We further sought the RP105 protein, which might have been produced but stayed inside the cells because of the lack of MD-1. We stained MD-1-deficient B cells that had been subjected to membrane-permeabilizing treatment with the aim to detect intracellular RP105, but we could not detect it (data not shown). We next treated B cells lacking MD-1 with recombinant soluble MD-1, which, however, did not complement cell surface RP105 expression (data not shown). Collectively, the coexpression of MD-1 is indispensable for cell surface RP105 expression in vivo. In the meantime, MD-1 was not detected on B cells lacking RP105 (Figure 6). MD-1 may be membrane-anchored only by RP105 on the B-cell surface.
Antagonistic effect of the anti-MD-1 monoclonal antibody on B-cell responses to lipopolysaccharide We further asked whether MD-1 contributed functionally to B-cell activation by antibody-mediated RP105 ligation or LPS. Anti-MD-1 mAbs were included in in vitro B-cell activation studies. We have established as many as 20 independent mAbs to RP105, all of which induced robust B-cell proliferation (Miyake et al14 and data not shown). In sharp contrast, none of the 9 mAbs to MD-1 was mitogenic (Figure 7 and data not shown). In this regard, all the anti-MD-1 mAbs we obtained were functionally distinct from the highly mitogenic anti-RP105 mAbs. Anti-MD-1 mAbs were included in B-cell proliferation driven by a variety of stimuli (Figure 7). They also were inhibitory of B-cell proliferation driven by LPS and by an anti-RP105 mAb, and they also suppressed LPS-induced up-regulation of B7.2 (Figure 8). Inhibition was specific because the anti-MD-1 mAb had no effect on the anti-IgM-induced B7.2 up-regulation. LPS-induced up-regulation of CD23 was barely influenced by the anti-MD-1 mAb, showing consistency with the results using B cells lacking MD-1.
RP105 does not interact with MD-2 Finally, we sought to determine whether RP105 interacts with MD-2. Ba/F3 cells expressing RP105 and MD-2 were established and used for immunoprecipitation with anti-RP105 mAb (Figure 9A). We could not, however, detect coprecipitation of MD-2, which was abundantly expressed as shown by immunoprecipitation and immunoprobing with the anti-flag mAb (Figure 9B, lane 2). The RP105 signal from Ba/F3 cells expressing RP105 and MD-2 was low compared with the RP105 signal from Ba/F3 cells expressing RP105/MD-1 (Figure 9A, lanes 1 and 2). Because cell surface RP105 is dependent on the coexpression of MD-1,12 we could not obtain Ba/F3 cells expressing MD-2 and RP105 whose expression is as high as on those expressing RP105/MD-1. This result is consistent with the results that cell surface RP105 is absent on MD-1-deficient B cells (Figure 6). To obtain higher expression of RP105 on the cell surface, we used Ba/F3 cells expressing RP105/MD-1 and TLR4/MD-2 (lane 4). RP105 was precipitated with anti-RP105 mAb, and coprecipitated MD-2, if there was any, was probed with anti-flag mAb. We still could not see MD-2 coprecipitation. Taken together with the absence of RP105 on B cells lacking MD-1 but expressing endogenous MD-2,11 RP105 was not likely to substitute MD-2 for MD-1.
The current study underscored a role for MD-1 in B-cell responsiveness to LPS by producing mice that lacked MD-1. MD-1-deficient mice are similar to RP105-deficient mice in B-cell phenotype. Both types showed hyporesponsiveness in LPS-induced B-cell proliferation and antibody production. This similarity between the 2 mutant mice is attributed to the lack of cell surface RP105/MD-1 (Figure 6). MD-1 is indispensable for cell surface expression of RP105. We previously established Ba/F3 cells expressing RP105 alone by transfecting the expression vector encoding RP105.12 Expression was enhanced by further transfection of MD-1. A role for MD-1 in vivo was found to be more critical than those in vitro results. Considering that RP105 is not expressed on B cells lacking MD-1 but expressing endogenous MD-2 (Figure 6 and 11), RP105 does not seem to substitute MD-2 for MD-1. In keeping with this, RP105 expression was low in Ba/F3 cells overexpressing RP105 and MD-2, and we could not see an association between RP105 and MD-2 (Figure 9). To achieve higher expression of RP105 and MD-2 on the cell surface, we also used Ba/F3 cells expressing RP105/MD-1 and TLR4/MD-2. We could not see the association between MD-2 and RP105 or RP105/MD-1 (Figure 9, lanes 3 and 4). These results revealed a specific interaction between RP105 and MD-1 but not with MD-2. The heavy chain of major histocompatibility complex (MHC) class I is
associated with Flow cytometry staining with cell membrane permeabilization did not detect intracellular RP105 in B cells lacking MD-1. Misfolded RP105 would not accumulate but is likely to be degraded. Even after the treatment of spleen cells lacking MD-1 with proteasome inhibitors such as lactacystin and N-carbobenzoxyl-leucinyl-leucinal,27 we could not detect intracellular RP105 by flow cytometry staining with cell membrane permeabilization. Additional biochemical studies using metabolic labeling and immunoprecipitation of RP105 would be required for revealing an intracellular fate of RP105 in the absence of MD-1. We established as many as 20 independent mAbs to RP105, all of which induced robust B-cell proliferation (Miyake et al14 and data not shown). In sharp contrast, none of the 9 anti-MD-1 mAbs was mitogenic (Figure 7 and data not shown). The anti-MD-1 mAbs should be similar to the anti-RP105 mAbs in cross-linking RP105/MD-1; nonetheless, only anti-RP105 mAbs are mitogenic. Cross-linking of RP105/MD-1 is insufficient for triggering B-cell activation. It must be noted that the cytoplasmic portion of RP105 consists of only 11 amino acids, which would be too short to deliver an activation signal. Another molecule may deliver an activation signal for RP105/MD-1. The anti-MD-1 mAbs might disrupt a link between RP105/MD-1 and such a putative signal transducer, whereas the anti-RP105 mAbs could strengthen the association and trigger an activation signal through the putative signal transducer. A search for the signal transducer is underway. Using anti-MD-1 mAbs, we showed a functional role for MD-1 in RP105-dependent and LPS-triggered B-cell activation. The anti-MD-1 mAbs were found to be antagonistic on LPS-induced proliferation and B7.2 up-regulation. These results are consistent with LPS hyporesponsiveness in mice lacking either RP105 or MD-1, and they suggest a functional role for MD-1 in B-cell responses to LPS. We believe that MD-1 is not just a molecule that helps RP105 to come out on the cell surface but that MD-1 helps RP105 to signal LPS, as MD-2 does TLR4. LPS-induced up-regulation of CD23 was not impaired in B cells lacking MD-1 (Figure 4). Anti-MD-1 mAb was unable to inhibit LPS-induced CD23 up-regulation (Figure 8). TLR4 is, on the other hand, indispensable for this response because B cells derived from TLR4-mutated C3H/HeJ mice do not show CD23 up-regulation in response to LPS (data not shown). CD23 up-regulation seems to require the LPS signaling through TLR4 but not through RP105/MD-1. In keeping with this, anti-RP105, antibody-dependent ligation of RP105/MD-1 did not lead to CD23 up-regulation (Figure 8B). These results reveal that LPS responses in B cells are not simple but are heterogeneous in their requirement for the signal through RP105/MD-1. B cells require the signal through RP105/MD-1 and also through TLR4/MD-2 for B7.2 up-regulation, proliferation, and antibody production, but not for CD23 up-regulation. It is important to note that tissue distribution of TLR4/MD-2 and RP105/MD-1 is not similar. TLR4/MD-2 is broadly expressed in organs including heart, kidney, brain, and liver,5,6,8 whereas RP105/MD-1 is restricted to immune cells such as B cells, macrophages, and dendritic cells.12,28 Nonimmune cells are likely to use only TLR4/MD-2 for LPS recognition and signaling. B cells, on the other hand, have an additional recognition or signaling pathway through RP105/MD-1. Gorczynski et al29 showed that MD-1 down-regulation with the antisense oligodeoxynucleotides led to impairment in LPS-induced CD80/CD86 up-regulation on bone marrow-derived dendritic cells. The antisense treatment is likely to result in the down-regulation of RP105/MD-1. Dendritic cells may be similar to B cells in their requirement for RP105/MD-1 in LPS recognition and signaling. Further studies about mice lacking MD-1 will focus on cell lineages other than B cells, including macrophages and dendritic cells.
Submitted May 15, 2001; accepted October 19, 2001.
Supported by Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government (Monbukagakusho); Uehara Memorial Foundation; Yamanouchi Foundation for Research on Metabolic Disorders; Mitsubishi-Tokyo Pharmaceutical; and Sankyo.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kensuke Miyake, Department of Microbiology and Immunology, Division of Infectious Genetics, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639; e-mail: kmiyake{at}ims.u-tokyo.ac.jp.
1. Janeway CA Jr. Approaching the asymptote: evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol. 1989;54:1-13. 2. Fearon DT, Locksley RM. The instructive role of innate immunity in the acquired immune response. Science. 1996;272:50-53[Abstract]. 3. Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature. 2000;406:782-787[CrossRef][Medline] [Order article via Infotrieve]. 4. Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol. 1995;13:437-457[CrossRef][Medline] [Order article via Infotrieve]. 5. Medzhitov R, Preston-Hurlburt P, Janeway CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997;388:394-397[CrossRef][Medline] [Order article via Infotrieve].
6.
Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF.
A family of human receptors structurally related to Drosophila Toll.
Proc Natl Acad Sci U S A.
1998;95:588-593
7.
Poltorak A, Xialong H, Sminova I, et al.
Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in tlr4 gene.
Science.
1998;282:2085-2088
8.
Qureshi ST, Lariviere L, Leveque G, et al.
Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4).
J Exp Med.
1999;189:615-625
9.
Hoshino K, Takeuchi O, Kawai T, et al.
Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the LPS gene product.
J Immunol.
1999;162:3749-3752 10. Arbour NC, Lorenz E, Schutte BC, et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet. 2000;25:187-191[CrossRef][Medline] [Order article via Infotrieve].
11.
Shimazu R, Akashi S, Ogata H, et al.
MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4.
J Exp Med.
1999;189:1777-1782
12.
Miyake K, Shimazu R, Kondo J, et al.
MD-1, a molecule that is physically associated with RP105 and positively regulates its expression.
J Immunol.
1998;161:1348-1353 13. Miyake K, Yamashita Y, Ogata M, Sudo T, Kimoto M. RP105, a novel B cell surface molecule implicated in B cell activation, is a member of the leucine-rich repeat protein family. J Immunol. 1995;154:3333-3340[Abstract]
14.
Miyake K, Yamashita Y, Hitoshi Y, Takatsu K, Kimoto M.
Murine B cell proliferation and protection from apoptosis with an antibody against a 105-kD molecule: unresponsiveness of X-linked immunodeficient B cells.
J Exp Med.
1994;180:1217-1224
15.
Miura Y, Shimazu R, Miyake K, et al.
RP105 is associated with MD-1 and transmits an activation signal in human B cells.
Blood.
1998;92:2815-2822 16. Roshak AK, Anderson KM, Holmes SD, et al. Anti-human RP105 sera induces lymphocyte proliferation. J Leukoc Biol. 1999;65:43-49[Abstract].
17.
Chan VWF, Mecklenbräuker I, Su I, et al.
The molecular mechanism of B cell activation by toll-like receptor protein RP105.
J Exp Med.
1998;188:93-101
18.
Ogata H, Su I, Miyake K, et al.
The Toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells.
J Exp Med.
2000;192:23-30
19.
Mizushima S, Nagata S.
pEF-BOS, a powerful mammalian expression vector [abstract].
Nucl Acid Res.
1990;18:5322 20. Palacios R, Steinmetz M. IL-3 dependent mouse clones that express B220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo. Cell. 1985;41:727-734[CrossRef][Medline] [Order article via Infotrieve].
21.
Horai R, Asano M, Sudo K, et al.
Production of mice deficient in genes for interleukin (IL)-1a, IL-1b, IL-1a/b, and IL-1 receptor antagonist shows that IL-1b is crucial in turpentine-induced fever development and glucocorticoid secretion.
J Exp Med.
1998;187:1463-1475 22. Veis DJ, Sentman CL, Bach EA, Korsmeyer SJ. Expression of the bcl-2 protein in murine and human thymocytes and in peripheral T lymphocytes. J Immunol. 1993;151:2546-2554[Abstract]. 23. Nomura J, Inui S, Yamasaki T, et al. Anti-CD40 monoclonal antibody induces the proliferation of murine B cells as a B-cell mitogen through a distinct pathway from receptors for antigens or lipopolysaccharide. Immunol Lett. 1995;45:195-203[CrossRef][Medline] [Order article via Infotrieve]. 24. Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of T cell costimulation. Annu Rev Immunol. 1996;14:233-258[CrossRef][Medline] [Order article via Infotrieve]. 25. Pamer E, Cresswell P. Mechanisms of MHC-class I-restricted antigen processing. Annu Rev Immunol. 1998;16:323-358[CrossRef][Medline] [Order article via Infotrieve]. 26. Lippincott-Schwartz J, Bonifacino JS, Yuan LC, Klausner RD. Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Cell. 1988;54:209-220[CrossRef][Medline] [Order article via Infotrieve]. 27. Kamhi-Nesher S, Shenkman M, Tolchinsky S, et al. A novel quality control compartment derived from the endoplasmic reticulum. Mol Biol Cell. 2001;10:1711-1723. 28. Fugier-Vivier I, de Bouteiller O, Guret C, et al. Molecular cloning of human RP105. Eur J Immunol. 1997;27:1824-1827[Medline] [Order article via Infotrieve].
29.
Gorczynski RM, Chen Z, Clark DA, et al.
Regulation of gene expression of murine MD-1 regulates subsequent T cell activation and cytokine production.
J Immunol.
2000;165:1925-1932
© 2002 by The American Society of Hematology.
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  T. Kobayashi, K. Takahashi, Y. Nagai, T. Shibata, M. Otani, S. Izui, S. Akira, Y. Gotoh, H. Kiyono, and K. Miyake Tonic B cell activation by Radioprotective105/MD-1 promotes disease progression in MRL/lpr mice Int. Immunol., July 1, 2008; 20(7): 881 - 891. [Abstract] [Full Text] [PDF]
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 A. Meyer-Bahlburg, S. Khim, and D. J. Rawlings B cell intrinsic TLR signals amplify but are not required for humoral immunity J. Exp. Med., December 24, 2007; 204(13): 3095 - 3101. [Abstract] [Full Text] [PDF]
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 |
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 S. Divanovic, A. Trompette, L. K. Petiniot, J. L. Allen, L. M. Flick, Y. Belkaid, R. Madan, J. J. Haky, and C. L. Karp Regulation of TLR4 signaling and the host interface with pathogens and danger: the role of RP105 J. Leukoc. Biol., August 1, 2007; 82(2): 265 - 271. [Abstract] [Full Text] [PDF]
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|
|
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Y. Wakabayashi, M. Kobayashi, S. Akashi-Takamura, N. Tanimura, K. Konno, K. Takahashi, T. Ishii, T. Mizutani, H. Iba, T. Kouro, et al. A Protein Associated with Toll-Like Receptor 4 (PRAT4A) Regulates Cell Surface Expression of TLR4 J. Immunol., August 1, 2006; 177(3): 1772 - 1779. [Abstract] [Full Text] [PDF]
|
|
|
|
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|
|
|
|
 |
|
 S. Divanovic, A. Trompette, S. F. Atabani, R. Madan, D. T. Golenbock, A. Visintin, R. W. Finberg, A. Tarakhovsky, S. N. Vogel, Y. Belkaid, et al. Inhibition of TLR-4/MD-2 signaling by RP105/MD-1 Innate Immunity, December 1, 2005; 11(6): 363 - 368. [Abstract] [PDF]
|
|
|
|
 |
|
 J. Koraha, N. Tsuneyoshi, M. Kimoto, J.-F. Gauchat, H. Nakatake, and K. Fukudome Comparison of Lipopolysaccharide-Binding Functions of CD14 and MD-2 Clin. Vaccine Immunol., November 1, 2005; 12(11): 1292 - 1297. [Abstract] [Full Text] [PDF]
|
|
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|
|
W. A. Derbigny, M. S. Kerr, and R. M. Johnson Pattern Recognition Molecules Activated by Chlamydia muridarum Infection of Cloned Murine Oviduct Epithelial Cell Lines J. Immunol., November 1, 2005; 175(9): 6065 - 6075. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Hebeis, E. Vigorito, D. Kovesdi, and M. Turner Vav proteins are required for B-lymphocyte responses to LPS Blood, July 15, 2005; 106(2): 635 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Nagai, T. Kobayashi, Y. Motoi, K. Ishiguro, S. Akashi, S.-i. Saitoh, Y. Kusumoto, T. Kaisho, S. Akira, M. Matsumoto, et al. The Radioprotective 105/MD-1 Complex Links TLR2 and TLR4/MD-2 in Antibody Response to Microbial Membranes J. Immunol., June 1, 2005; 174(11): 7043 - 7049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. B. Bekeredjian-Ding, M. Wagner, V. Hornung, T. Giese, M. Schnurr, S. Endres, and G. Hartmann Plasmacytoid Dendritic Cells Control TLR7 Sensitivity of Naive B Cells via Type I IFN J. Immunol., April 1, 2005; 174(7): 4043 - 4050. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Tsuneyoshi, K. Fukudome, J. Kohara, R. Tomimasu, J.-F. Gauchat, H. Nakatake, and M. Kimoto The Functional and Structural Properties of MD-2 Required for Lipopolysaccharide Binding Are Absent in MD-1 J. Immunol., January 1, 2005; 174(1): 340 - 344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Pugin, S. Stern-Voeffray, B. Daubeuf, M. A. Matthay, G. Elson, and I. Dunn-Siegrist Soluble MD-2 activity in plasma from patients with severe sepsis and septic shock Blood, December 15, 2004; 104(13): 4071 - 4079. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fujimoto, S. Yamazaki, A. Eto-Kimura, K. Takeshige, and T. Muta The Amino-terminal Region of Toll-like Receptor 4 Is Essential for Binding to MD-2 and Receptor Translocation to the Cell Surface J. Biol. Chem., November 12, 2004; 279(46): 47431 - 47437. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wagner, H. Poeck, B. Jahrsdoerfer, S. Rothenfusser, D. Prell, B. Bohle, E. Tuma, T. Giese, J. W. Ellwart, S. Endres, et al. IL-12p70-Dependent Th1 Induction by Human B Cells Requires Combined Activation with CD40 Ligand and CpG DNA J. Immunol., January 15, 2004; 172(2): 954 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Gioannini, A. Teghanemt, K. A. Zarember, and J. P. Weiss Regulation of interactions of endotoxin with host cells Innate Immunity, December 1, 2003; 9(6): 401 - 408. [Abstract] [PDF]
|
|
|
|
|
|
G. Meng, A. Grabiec, M. Vallon, B. Ebe, S. Hampel, W. Bessler, H. Wagner, and C. J. Kirschning Cellular Recognition of Tri-/Di-palmitoylated Peptides Is Independent from a Domain Encompassing the N-terminal Seven Leucine-rich Repeat (LRR)/LRR-like Motifs of TLR2 J. Biol. Chem., October 10, 2003; 278(41): 39822 - 39829. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yazawa, M. Fujimoto, S. Sato, K. Miyake, N. Asano, Y. Nagai, O. Takeuchi, K. Takeda, H. Okochi, S. Akira, et al. CD19 regulates innate immunity by the toll-like receptor RP105 signaling in B lymphocytes Blood, August 15, 2003; 102(4): 1374 - 1380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ichiba, T. Teshima, R. Kuick, D. E. Misek, C. Liu, Y. Takada, Y. Maeda, P. Reddy, D. L. Williams, S. M. Hanash, et al. Early changes in gene expression profiles of hepatic GVHD uncovered by oligonucleotide microarrays Blood, July 15, 2003; 102(2): 763 - 771. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Van Amersfoort, T. J. C. Van Berkel, and J. Kuiper Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clin. Microbiol. Rev., July 1, 2003; 16(3): 379 - 414. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 A K Singh and Y Jiang Lipopolysaccharide (LPS) induced activation of the immune system in control rats and rats chronically exposed to a low level of the organothiophosphate insecticide, acephate Toxicology and Industrial Health, March 1, 2003; 19(2-6): 93 - 108. [Abstract] [PDF]
|
|
|
|
|
|
K. Miyake, Y. Nagai, S. Akashi, M. Nagafuku, M. Ogata, and A. Kosugi Essential role of MD-2 in B-cell responses to lipopolysaccharide and Toll-like receptor 4 distribution Innate Immunity, December 1, 2002; 8(6): 449 - 452. [Abstract] [PDF]
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Top
Abstract
Introduction
) and
MD113 (rat IgG2b/
/
) or
MD-1-deficient mice (
) were stimulated for 3 days with
anti-CD40 (10 µg/mL); anti-RP105 (2 µg/mL); lipid A (0.01, 0.1, and
1.0 µg/mL); LPS from S minnesota Re595 or E
coli 055: B5 (0.01, 0.1, and 1.0 µg/mL). [3H]-TdR
uptake was counted as described in "Materials and methods." Results
were shown as mean values of count per minute (cpm) from triplicate
wells with standard errors. When compared with wild-type B cells,
MD-1-deficient B cells showed significant hyporesponsiveness to LPS as
revealed by the following P values: lipid A 0.01 µg/mL,
P < .001; lipid A 0.1 µg/mL, P < .002;
lipid A 1.0 µg/mL, P < .003; Re595 0.01 µg/mL,
P < .011; Re595 0.1 µg/mL, P < .001;
Re595 1.0 µg/mL, P < .0001; 055:B5 0.01 µg/mL,
P < .11; 055:B5, 0.1 µg/mL, P < .002;
055:B5 1.0 µg/mL, P < .001.
) and MD-1-deficient mice (
) were immunized
intraperitoneally with TNP-LPS (20 µg per mouse) and bled 1 and 2 weeks later. Five mice were used for each group. Titers of TNP-specific
IgM or IgG3 were measured with ELISA as described in "Materials and
methods."
2-microglobulin
(