Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines

Steven E. Applequist1, Robert P. A. Wallin1 and Hans-Gustaf Ljunggren1,2

1 Microbiology and Tumor Biology Center, Karolinska Institutet, 171 77 Stockholm, Sweden 2 Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Huddinge University Hospital, 141 86 Stockholm, Sweden

Correspondence to: S. Applequist; E-mail: steven.applequist{at}mtc.ki.se
Transmitting editor: H. L. Ploegh


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Pattern recognition receptors (PRR) play an important roll in immediate responses to different conserved molecules produced by microbes. In this paper we describe the cloning of the mouse homolog of Toll-like receptor (TLR) 3, and present an analysis of the expression of this gene in innate and adaptive immune cell lines. We also performed a broad expression study on these cells of other TLR, including TLR family members whose expression pattern is not known, i.e. TLR7. The analysis was done in order to understand, and possibly predict, how innate and adaptive immune cells respond to microbial pattern antigens. This first large-scale analysis of immune cell TLR expression in the mouse reveals that cells of the innate immune system express a broader number of TLR than cells of the adaptive immune system, supporting preconceptions concerning the hierarchy of immune cells involved in direct pathogen recognition. Additionally, the expression of TLR transcripts by mast cells, neutrophils and microglial cells observed here suggests that pathogen-associated molecular pattern molecules could induce activation of these cells through TLR. Finally, the mouse homolog of human TLR3 identified here may, like its human counterpart, be an exceptional TLR molecule due to its lack of a conserved proline residue seen to be involved in existing TLR signaling capabilities found in other TLR family members.

Keywords: cellular activation, inflammation, innate immunity, mouse


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Immunity to infections by microorganisms is carried out by a large array of weapons that are triggered by pathogen encounter. Direct and immediate recognition of pathogens can be mediated by a set of germline-encoded receptors which are referred to as pattern recognition receptors (PRR) which recognize molecular determinants specific to certain classes of pathogens. These determinants are often referred to as a pathogen-associated molecular pattern molecules (PAMP) (1). One class of PRR are the Toll and the Toll-like receptors (TLR) which consist of a family of transmembrane molecules found in a broad range of organisms from Drosophila to higher mammals (24), and play a roll in immune responses to a variety of microbial pathogens and their constituents (57). TLR are characterized by an extracellular domain consisting of multiple leucine-rich repeat elements and a cytoplasmic domain that belongs to the IL-1/TOLL receptor family which is able to transduce signals through MyD88, IRAK, NF-{kappa}B and mitogen-activated protein kinase signaling cascade (6) leading to anti-microbial immune responses. PAMP can activate cells through TLR family members which can lead to a wide variety of responses from different cell types ranging from chemokine (814) and cytokine production (1526), increases in antigen-presentation capacity (6,16,19,2729), co-stimulatory (2,6,19,28,29) and adhesion molecules (28), initiation of cell proliferation (16,19,29,30), and antimicrobial factor production (12,21, 27,3137). Additionally, observations that cooperation between different TLR molecules leads to recognition of PAMP (11,12,38,39) allows even greater variability in microbial recognition using the limited number of TLR family members. These observations reinforce the importance of knowing which TLR molecules a cell expresses as well as accessory molecules such as CD14 and key TLR signaling molecules such as MyD88 (4,40). Currently, there is only limited information on the expression of these receptors by mouse cells. Most studies published primarily focus on the expression of a limited number of TLR family members in few cell types. The aim of the present work was to identify new murine TLR family members (to increase the number of TLR that could be analyzed) and to determine which cell types they, and other previously known TLR, are expressed by. This study was done to better understand the overall structure of the TLR defense system in cells of the immune system and to form a foundation for the possible prediction of which immune cell types may be dominant in TLR-dependent responses to certain microorganism PAMP. Here we have studied the extent of expression of eight murine TLR, CD14 and MyD88 mRNA transcripts in 17 different cell lines representing innate and adaptive immune cells of both lymphoid and myeloid origin.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Mice
BALB/c, C57BL/6 and DBA mice were housed under standard specific pathogen-free conditions at the Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm. All procedures were performed under both institutional and national guidelines.

Cloning of murine TLR3
Human TLR sequences were compared to the murine expressed sequence tag (mEST) database using the BLAST (http://www.ncbi.nih.gov/BLAST/) database (41). TLR3 was identified and cloned using the mEST IMAGE. Consortium (42) [LLNL (http://image.llnl.gov/)] cDNA Clone (accession no. AA980131). The 5' end was captured using a primer based on the incomplete 3' cDNA clone mEST sequence and the Smart Race cDNA amplification Kit (Clonetech, Palo Alto, CA) according to the manufacturer’s instructions. Using sequence information from the mEST and 5'-RACE products, cDNA fragments primers were designed to isolate a clone encoding the complete open reading frame from J774.1 cDNA (TLR3-5' 5'-TAGAATATGATACAGGGATTGC-3', TLR3-3' 5'-TTACCTC AAGTAGCCTTATACC-3') using the Expand Long Template PCR system (Roche Diagnostics Scandinavia, Bromma, Sweden) of which four clones were isolated, sequenced and the consensus deposited as GenBank no. AF355152. TLR7 was identified and cloned using the mEST clone (accession no. AW476242) in a manner similar to TLR3 as described above. The clones sequence was 100% identical to a recently deposited mTLR7 clone (accession no. AY035889; F. J. Heil et al., unpublished).

RNA isolation and cDNA preparations
Total RNA was isolated from cell lines, purified cells or whole tissue using the TRIzol isolation solution according to the manufacturer (Life Technologies, Rockville, MD). Isolated RNA was treated with RNase-free DNase I using the DNA-free kit (Ambion, Austin, TX). Absence of DNA contamination was confirmed using the ß-actin primer pair and conditions described below for 35 cycles. cDNA was prepared using the First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. Briefly, 3 µg of total RNA was used as template in 33 µl cDNA reactions primed using an oligo(dT) primer for 1 h at 37°C.

PCR reactions and primers
A ß-actin primer pair mB-actin F (5'-GTG GGG CGC CCC AGG CAC CA-3') and mB-actin R (5'-CTCCTTAATGTCA CGCACGAT-3') were used as negative and positive controls for genomic DNA contamination of RNA and presence of cDNA respectively. PCR analyses of various TLR were done using the following primer pairs: TLR1, 5'-AGACAGCAC AAAGATGG-3' and 5'-TCCCCAAAGTTACATATAC-3'; TLR2, 5'-AGCTCTTTGGCTCTT CTG-3' and 5'-AGAACTGGGGG ATATGC-3'; TLR3, 5'-CAGTTCAGAAAGAACGG-3' and 5'-AGCCTTATACCATAAAAGC-3'; TLR4, 5'-GCATGGCTTACA CCACCTCT-3' and 5'-GTGCTGAAAATCCAGGTGCT-3'; TL5, 5'-GCAGATAATTCCTGAAGG-3' and 5'-CTTCCTTTAAAATG CATCCG-3'; TLR6, 5'-AATGACTTTGATGTACTG-3'; TLR7, 5'-AGAAACCTCCAGGAAC-3' and 5'-CATACACAATAAAAGC ATC-3'; TLR9, 5'-TCCCTGTATAGAATGTG-3' and 5'-TGGA GGCGTGAGAG-3'; CD14 5'-CTAGTCGGATTCTATTCGGA GC-3' and 5'-AGACAGGTCTAAGGTGGAGAGG-3' (43); MyD88 5'-ATCCGAGAGCTGGAAACG-3' and 5'-GCAAGGG TTGGTATAATC-3' (44). All PCR reactions were done in the presence of 1 mM dNTPs (Life Technologies), 2 µM MgCl, 1 x PCR buffer (Life Technologies), 2 U Taq DNA polymerase (Life Technologies), 20 µM of each primer and 1 µl of cDNA (90 ng RNA equivalent) prepared as described above in a total volume of 50 µl. All reactions were temperature cycled at 96°C/1 min, 54°C/1 min, 72°C/1.5 min for 30 cycles with the exception of TLR4, CD14 and MyD88, which were cycled at 96°C/1 min, 61°C/1 min, 72°C/1.5 min for 30 cycles. To control for polymorphism, all TLR primer pairs were confirmed to work either on cell lines derived from all three mouse strains and/or on tissue(s) from the strains of mice themselves using the conditions described here, ensuring that the lack of signal in a cell line is not due to the inability of primers to bind and amplify their target cDNAs.

Tissues, cells and cell lines
The following cell lines were used: fetal skin dendritic cells (FSDC) (45), CB-1 (46), D2SC/1 (47), RAW 264.7 (48), J774.1 (49-51), SV40 MES-13 (52), BV-2b (53), MCP5/L (54,55), IC-2 (56), WEHI 231 (57), 70Z/3 (58), J558 (59), S194 (60), RMA (61), ALC (62), CTLL-2 (63) and 32D (64,65). All cell lines were all grown in RPMI 1640 medium with the addition of 5–7.5% heat-inactivated FCS (Life Technologies), 2 mM L-glutamine (Life Technologies), penicillin/streptomycin (Life Techno logies) and 50 µM ß-mercaptoethanol (Sigma, St Louis, MO), with the exception of IC-2 and MCP5/L, which were grown in the above medium with the addition of 10% IL-3-conditioned media derived from the IL-3-producing murine cell line WEHI-B3. The cell line CTLL-2 was grown in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES and 1.0 mM sodium pyruvate, and supplemented with an additional 2 mM L-glutamine and 10 U/ml recombinant human IL-2 (PeproTech, Rocky Hill, NJ). All adherent cell lines were grown to a density of ~75% before RNA isolation. Bone marrow-derived DC were obtained from BALB/c mice using the protocol of Inaba et al. (66). The cells were harvested after 6 days, replated overnight, harvested at day 7 and total RNA was isolated. All mouse tissues were isolated from untreated mice, immediately frozen in liquid nitrogen and stored at –70°C before total RNA extraction.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
A growing number of PRR receptors belonging to the TLR family are being identified, but the knowledge of which cells express these receptors is still limited. Here we have cloned a new member of the TLR family in mouse and have analyzed its, and existing TLR members, expression on innate and adaptive immune cells.

Identification of murine orthologs of human TLR cDNAs
To identify murine orthologs of human TLR genes we used a comparative bioinformatics approach. When searching the mEST database of cDNA clones with the complete human TLR3 cDNA sequences, many independent mEST were identified. mEST clones with the highest matching base-pair identities were obtained, sequenced and the information was used to capture complete cDNA clones from the murine macrophage cell line J774.1. For TLR3 four clones were isolated and sequenced, and from them a consensus sequence was derived. The TLR3 3310 nucleotide sequence contains an open reading frame from nucleotides 44 to 2761. The ATG codon of TLR3 at nucleotide 44 is very likely the true translation initiation codon because it is preceded by two in-frame TGA stop codons at nucleotide 8 and 33. TLR3 also contains motifs associated with the TLR family such as leucine-rich repeats in its extracellular domain as well as a Toll/IL-1 receptor homology domain in its cytoplasmic tail. The predicted amino acid sequence of TLR3 showed 79% identity and 87% similarity at the amino acid level to that of human TLR3 as determined using the BLAST 2 sequences program (Fig. 1). These values are within the range of similarities and identities seen between other human and mouse TLR molecules (data not shown) shown to be both molecular orthologs [TLR5 (3,67) and 9 (19,68)] as well as functional homologs [TLR1 (11,38,39), 2 (16,69,70), 4 (2,27,71) and 6 (25)], suggesting that mouse TLR3 described here indeed encodes the murine ortholog of human TLR3. It has been seen that the ability of TLR molecules to signal requires a conserved proline residue found within the cytoplasmic tail of the TLR family (6). This residue in the so-called BB loop is essential in recruiting a key TLR signaling adaptor molecule MyD88 (72), but does not affect interactions with the signaling adaptor molecule TIRAP (73). This proline residue is also changed to a histidine in the TLR4 mutant mouse C3H/HeJ which is lipopolysaccharide (LPS) hyporesponsive (27,71), and has also been changed from proline to histidine in engineered versions of dominant-negative TLR1, 2 and 6 molecules in other systems (38,39) demonstrating it is a critical residue involved in TLR function. Interestingly, the predicted amino acid sequence of mouse TLR3 lacks a proline residue at amino acid 795 which is also absent at position 794 in human TLR3 [also observed by Kaisho and Akira in the putative mouse TLR3 EST clone, AA980131 (6)]. The absence of this residue in both the human TLR3 (3) as well as the complete mouse TLR3 identified here confirms that TLR3 is an exceptional molecule within the TLR family. While in submission, the ligand and signaling capabilities of TLR3 have been identified. Apparently, double-stranded RNA is a molecule able to activate cells expressing TLR3 using both MyD88-dependent and -independent mechanisms (74), suggesting that the conserved proline in other TLR may not be as directly responsible for TLR function as once believed.



View larger version (86K):
[in this window]
[in a new window]
 
Fig. 1. Comparison of the predicted amino acid sequences of human and murine TLR3. Entire deduced amino acid sequences of human TLR3 and comparison with the mouse TLR3 amino acid sequence are shown. The numbers on the left indicate amino acid positions. Identical amino acid residues are shaded gray. A lower case character represents a conserved amino acid difference in the murine sequence when compared to that of the predicted human sequence as determined by the BLASTp alignment program. The predicted signal peptides (TLR3 human: residues 1–23; TLR3 mouse: residues 1–24) are underlined. The predicted transmembrane segments (human: residues 704–725; mouse: residues 705–726). The position of the conserved functional amino acid residue within TLR cytoplasmic tails is underlined and in bold. Nucleotide sequences encoding the deduced murine TLR3 amino acid sequences shown have been deposited with GenBank; murine TLR3 (accession no. AF355152).

 
Expression of TLR, CD14 and MyD88 in various murine lymphoid and myeloid cell lines
Expression of a panel of 10 cDNAs encoding eight TLR, CD14 and MyD88 were analyzed in murine lymphoid and myeloid cell lines using RT-PCR. Cell lines representing the innate immune system were immature DC (CB-1, D2SC/1 and FSDC), macrophages from different tissue sources (J774.1, RAW 264.7 and SV40 MES-13), a microglial cell line (BV-2b), mast (MCP5/L) and mast/basophil-like (IC-2) cells, and the granulocyte macrophage colony stimulating factor (G-CSF)-differentiated cell line 32D representing neutrophilic granulocytes. Cell lines representing the adaptive arm of the immune system were cells of the immature B (70Z/3 and WEHI231) and plasma cell lineage (S194 and J558) as well as a T cell line (CTLL-2) and T cell lymphomas (RMA and ALC).

Results are shown in Fig. 2 and summarized in Table 1. TLR1, 2, 3, 4 and 9 mRNA transcripts are expressed in cell lines derived from the three mouse strains used (BALB/c, C57BL/6, DBA/2 and C3H/HeJ), while the ability to detect TLR5, 6 and 7 mRNA transcripts using the indicated primer pairs was confirmed by using cDNA reverse transcribed from liver and skin (TLR5) and spleen (TLR6 and 7) from the three mouse strains used (data not shown). RT-PCR products were of expected size and confirmed by sequence analysis (data not shown).



View larger version (111K):
[in this window]
[in a new window]
 
Fig. 2. Expression of TLR in murine lymphoid and myeloid cell lines. Total RNA was isolated from the indicated cell lines and first-strand cDNA synthesis was performed as indicated in Methods, and subjected to PCR amplification. Products were resolved using agarose gel electrophoresis, stained with ethidium bromide and photographed. m{phi} = macrophages, B = B cells, T = T cells, MB = mast cells/basophil cells, G/Nu = granulocytic/neutrophil cells, (+) = positive controls were whole spleen cDNA for all reactions with the exception of TLR5 where skin cDNA was used and TLR6 where bone marrow derived DC were used, (–) = negative control. Data are representative of two to three independent amplification experiments done using one to two independent reverse transcription reactions with similar qualitative results. The ß-actin control shown is representative of product amplification in all PCR experiments performed. The additional band seen in TLR4 PCR amplification reactions is also TLR4 as confirmed by DNA sequence analysis (data not shown).

 

View this table:
[in this window]
[in a new window]
 
Table 1. Summary of gene expression in murine lymphoid and myeloid derived cell linesa
 
Analysis of TLR expression is a difficult issue. It has been found that TLR function correlates better with mRNA expression than with cell surface detection of TLR protein with mAb. Highly LPS-responsive human immature DC express very low levels of TLR4 molecules which are detectable by northern blot but not by antibody staining (75). This suggests that the use of antibodies to detect functional TLR expression may not always be sensitive enough. RT-PCR is a sensitive technique, but might detect rare transcripts originating from contaminating cells in ex vivo purified cell populations. To avoid this problem we used tissue culture cell lines or, in the case of the cell line 32D, a heterogeneous cell population derived from a differentiated homogenous cell line. Using RT-PCR data to draw conclusions about protein expression is always speculative; however, in the absence of antibodies to study protein expression, it is a informative and an important first-step analysis technique.

The expression analysis of recently identified mouse TLR3 as well as known mTLR transcripts and the new mouse TLR7 reveals interesting expression patterns. First, there are TLR molecules expressed to some degree in all classes of cells tested, TLR2, 4 and 6. Second, TLR1 is expressed primarily in cells classically thought to be involved in innate and immediate immune responses. Thirdly, TLR5 appears to be restricted to a subset of innate cells representing only DC and (select) macrophages. Finally, we see a correlation where cells that express TLR7 also express TLR9 transcripts. We also find expression of TLR3 in a variety of cell types which differs from a study using purified human cell populations showing exclusive TLR3 expression in monocyte-derived DC (76). This may be due to the different analysis technique used or that mouse TLR3 is expressed in a different population of cells compared to humans.

The predominant expression of TLR by the innate immune cell lines studied here indicates that they likely represent a class of instructive cells which are able respond to a large number of PAMP and are capable of influencing the activities of other immune cells. Additionally, it supports the preconception that innate immune cells play a dominant roll in the first line of microbial defense.

TLR expression by DC lines
DC are a cell type highly responsive to microbial antigens such as LPS, LTA and CpG DNA, and indeed they have been shown to express TLR4 (7577), 2 (28,7577), 6 (75) and 9 (19) as well as other TLR (75,76). Analysis of three DC lines revealed two classes of TLR expression. FSDC, which has been shown to represent a macrophage/immature DC-like surface phenotype (45), and the line D2SC/1, a splenic-derived DC cell line, express every TLR transcript analyzed at easily detectable levels, as well as CD14 and MyD88, suggesting that they represent an immature DC cell phenotype capable of responding to many classes of PAMP, similar to what has been suggested to represent the function of DC in vivo (78). They also produce iNOS (45), tumor necrosis factor (TNF)-{alpha} and IL-1ß in response to LPS (45,47), suggesting that the CD14 and TLR4 they express is functional. CB1 cells which are also derived from total spleen and have a DC phenotype (46) were found to express MyD88 and CD14 but lower levels, or limited numbers, of TLR transcripts. They do, however, express TLR4 and CD14 which coincides with their ability to produce TNF-{alpha} and IL-1ß in response to stimulation by LPS (46). It has been suggested however that they are lymphoid-derived DC (46) rather than myeloid derived (78). A difference that could be reflected in TLR expression. It is clear, however, that the DC cell lines analyzed here all express MyD88, CD14 and a large number of TLR, indicating that they represent a cell population which likely responds to a large variety of PAMP.

TLR expression by macrophage and macrophage-like cell lines
The macrophage and microglial cell lines analyzed here were selected for two reasons. First, they represent histologically similar, endocytic cells of the macrophage or macrophage-like lineage which we predicted would express similar TLR expression patterns due to the ability of macrophages to recognize and respond to PAMP (4). Second, we also wanted to study macrophage subsets such as reticular or mesangial cells represented by peritoneal or kidney-derived macrophage lines respectively. Analysis of three of the macrophage lines shows a number of similarities. They all express MyD88, CD14 and a large variety of TLR ranging from TLR1 to 9 similar to DC cell lines, indicating that they also represent a cell phenotype capable of responding to a large variety of PAMP. Additionally, we find that these cell lines express different TLR depending on where they were originally derived from. Macrophage cell lines of reticular phenotype (J774.1 and RAW 264.7) appear to express similar TLR transcripts (TLR5 excepted), whereas the macrophage cell line SV40 MES-13, derived from glomerular mesangial cells, appears to express many TLR, but appears to be different in that there is limited detection of TLR1, 6 and 7, and absence of TLR9. It is interesting to speculate that macrophages residing in different parts of the body may express different TLR receptors depending on the types of pattern antigens they are likely to encounter. However, it is not known whether these observed differences in TLR expression represent actual expression differences expressed by different macrophage subsets or represent differences in cellular expression at the time of transformation. BV-2b, a microglial cell line, which is similar to traditional macrophages and responds to LPS (53), appears to depart from the TLR expression seen above. Surprisingly, it expressed only TLR1, 4, 7 and 9 transcripts, as well as MyD88 and CD14, some of which are receptors involved in responses to LPS (4) and CpG DNA (19), suggesting that microglial cells may have a limited ability to respond to PAMP through TLR activation compared to traditional macrophages.

TLR expression by mast cell lines
It is known that mast cells are able to direct innate immune responses against bacterial infections in vivo (79,80), release pre-stored TNF-{alpha} in response to whole bacteria (79,81,82), and also produce the potent inflammatory cytokines IL-6, IL-9 and IL-13 in response to in vitro LPS stimulation (24,83). It has also been observed that they express RNA transcripts for TLR4 and CD14 (24), and TLR2, 4, 6 and 8 (26). We were also able to detect the presence of TLR2, 4 and 6, and CD14 transcripts in our mast cell lines but also found TLR1, suggesting that mast cells may also respond to PAMP using this regulatory TLR as well (39). Interestingly, we did not detect TLR7 or 9, which breaks from the pattern established by the other innate immune cell lines seen here. It has been recently shown that TLR4 expression by mast cells plays a roll in anti-microbial defenses (26); however, whether other TLR expressed by mast cells are also important in defense mechanisms is not known. Nevertheless, the identification of additional TLR molecules expressed by mast cell lines shown here suggests that their in vivo counterparts may have the ability to respond to a variety of additional PAMP and to influence innate immune responses.

TLR expression by a neutrophil cell line
Neutrophils play an important part in inflammatory reactions by responding to signals such as TNF-{alpha} and posses the ability to release a barrage of factors able to directly kill a large array of different microorganisms (84,85). Unfortunately, the expression of TLR by this highly effective cell population has not been extensively studied. The removal of IL-3 and addition of G-CSF to the cell line 32D leads to its differentiation from a proliferating myelocytic blast cell towards non-proliferating segmented neutrophilic granulocytes (65). We found that these potent cells expressed MyD88, CD14 as well as TLR1, 2, 3, 4, 6, 7 and 9 transcripts in cells treated with G-CSF for 6 days. The expression of CD14 and TLR4 by these cells is not unexpected as treatment of freshly derived neutrophils with LPS leads to priming as well as superoxide release and 5-lipoxygenase products in a CD14-dependent manner (86) and also increases their ability to effect a respiratory burst (87). The identification of TLR transcripts expression by this neutrophil line suggests that they are able to respond to a large variety of PAMP using the TLR system.

TLR expression by B cells
As representatives of the B cell arm of the adaptive immune system we used the immature sIgM µ+ {kappa}+ pre-B cell lymphoma 70Z/3, the sIgM+ B lymphoma WEHI 231 and the Ig-secreting B cell plasmacytomas S194 and J558. We found that the immature B cells expressed MyD88, TLR2, 3, 4, 6 and 9, while the plasmacytomas S194 and J558 expressed predominantly only MyD88, TLR2, 4 and 6. The expression of TLR4 by 70Z/3 cells is supported by earlier observations that they secrete IgM upon LPS stimulation (58). Expression of TLR2, 4 and 6 by WEHI 231 cells suggests that the rescue of anti-IgM-induced growth arrest by LPS and peptidoglycan (88) may be due to the expression of these receptors. It is not known, however, if the B cell lines studied here can be activated by other PAMP able to stimulate these TLR. It has been shown that mature resting B cells can be stimulated ex vivo by CpG DNA, a TLR9 ligand (89,90), and by the well-known B cell mitogens LPS and peptidoglycan. The lack of CD14 expression on the B cell lines observed here also supports other observations that the surface expression of CD14 by B cells is only seen in exceptional cases (43) and that the ability of B cells to respond to LPS may involve soluble CD14 (91). It is, however, not known if this pathway involves TLR molecules and/or recently identified receptors for LPS (92).

TLR expression by T cells
Resting T cells are refractory to stimulation by LPS (93,94) and CpG DNA (7), and therefore it was not surprising to find that the TCR+, CD3+ T cell lines RMA, ALC and the non-lymphoma cytotoxic T cell line CTLL-2 expressed at the limit of detection or non-detectable levels of TLR4 and 9 as well as all the other TLR studied here. It has, however, been seen that murine ex vivo {gamma}{delta} T cells express TLR4 and {alpha}ß T cells express TLR2 and 4 (2,95,96), suggesting that they may have the capacity to respond to PAMP. Nevertheless, the limited or non-detectable expression of many TLR transcripts by the T cell lines studied here suggests that in vivo, T cells could receive their instructions more from professional antigen-presenting cells rather than directly sensing and responding to their environment using TLR. The analysis of these B and T cell lines clearly demarcates a difference between TLR expression patterns and levels seen between these cell lines of the innate immune system and those of the adaptive system.

Possible determinants governing TLR expression
It is interesting that all cell types studied here express TLR receptors to a large degree or configuration with the general exception of T cells. It may be that the cell types that express a broader pattern of and greater levels of TLR have (i) the ability to produce a product which is able to directly play a part in responding to the microorganism and/or (ii) are capable of presenting antigen on MHC as well as co-stimulatory molecules CD80 (2,28) and CD86 (28). DC, macrophages, mast cells, neutrophils and even B cells are all able to present antigen under normal or cytokine-treated conditions, up-regulate co-stimulatory molecules, and secrete factors that can directly mediate microbial destruction. Therefore it is not surprising that all these cell types would posses a system which could immediately sense the presence of a microorganism and then monitor progress of its own work using TLR. Generally, classical {alpha}ß T cells (the lines studied here), on the other hand, are not in a position to make products that can directly affect the destruction of microorganisms or to act as an antigen-presenting cell. They better serve the immune response by receiving instructions from antigen-presenting cells and responding appropriately. It has been observed, however, that {gamma}{delta} T cells express TLR molecules according to RT-PCR performed on ex vivo sorted cells (96), and in mouse and human cell lines (2). This observation is not surprising, however, since they are thought to be more ‘innate’-like in there detection and responses to antigen. The further study of TLR receptors on T cells and their subpopulations should reveal the extent of control these receptors may or may not have on T cell activities.

The identification of TLR expression patterns by these immune cell lines suggests that in vivo expression of TLR transcripts by corresponding cells may be similar. If so, it may establish the cells lines described here as important models in which to study TLR responses.


    Acknowledgements
 
We thank Dr G. Nilsson for mast cell lines, Dr K. Bedecs for BV-2b, Dr M. J. Wick for CTLL-2, Dr T. Leanderson and E. Miller for WEHI 231 and J558, Dr E. Severinsson for 70Z/3 and S194, S. Erikson for J774.1, RAW 264.7 and MES-13 cells, and J-I. Jonsson for 32D RNA. We also thank H. Lönnquist for DNA sequencing support. This work was supported by a grant from the Swedish Medical Research Council, the Swedish Cancer Society, the Tobias Foundation and the Karolinska Institutet.


    Abbreviations
 
DC—dendritic cell

FSDC—fetal skin dendritic cell

G-CSF—granulocyte colony stimulating factor

LPS—lipopolysaccharide

mEST—murine expressed sequence tag

PAMP—pathogen-associated molecular pattern molecules

PRR—pattern recognition receptors

TLR—Toll-like receptor

TNF—tumor necrosis factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

  1. Medzhitov, R. and Janeway, C., Jr. 2000. Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173:89.[ISI][Medline]
  2. Medzhitov, R., Preston-Hurlburt, P. and Janeway, C. A., Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394.[ISI][Medline]
  3. Rock, F. L., Hardiman, G., Timans, J. C., Kastelein, R. A. and Bazan, J. F. 1998. A family of human receptors structurally related to Drosophila Toll. Proc. Natl Acad. Sci. USA 95:588.[Abstract/Free Full Text]
  4. Brightbill, H. D. and Modlin, R. L. 2000. Toll-like receptors: molecular mechanisms of the mammalian immune response. Immunology 101:1.[ISI]
  5. Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett, D. R., Eng, J. K., Akira, S., Underhill, D. M. and Aderem, A. 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099.[ISI][Medline]
  6. Kaisho, T. and Akira, S. 2001. Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice. Trends Immunol. 22:78.[ISI][Medline]
  7. Wagner, H. 2001. Toll meets bacterial CpG-DNA. Immunity 14:499.[ISI][Medline]
  8. Jeannin, P., Renno, T., Goetsch, L., Miconnet, I., Aubry, J. P., Delneste, Y., Herbault, N., Baussant, T., Magistrelli, G., Soulas, C., Romero, P., Cerottini, J. C. and Bonnefoy, J. Y. 2000. OmpA targets dendritic cells, induces their maturation and delivers antigen into the MHC class I presentation pathway. Nat. Immunol. 1:502.[ISI][Medline]
  9. Tapping, R. I., Akashi, S., Miyake, K., Godowski, P. J. and Tobias, P. S. 2000. Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides. J. Immunol. 165:5780.[Abstract/Free Full Text]
  10. Wang, M. J., Jeng, K. C. and Shih, P. C. 2000. Differential expression and regulation of macrophage inflammatory protein (MIP)-1alpha and MIP-2 genes by alveolar and peritoneal macrophages in LPS-hyporesponsive C3H/HeJ mice. Cell. Immunol. 204:88.[ISI][Medline]
  11. Wyllie, D. H., Kiss-Toth, E., Visintin, A., Smith, S. C., Boussouf, S., Segal, D. M., Duff, G. W. and Dower, S. K. 2000. Evidence for an accessory protein function for Toll-like receptor 1 in anti-bacterial responses. J. Immunol. 165:7125.[Abstract/Free Full Text]
  12. Bulut, Y., Faure, E., Thomas, L., Equils, O. and Arditi, M. 2001. Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein: role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling. J. Immunol. 167:987.[Abstract/Free Full Text]
  13. Frendeus, B., Wachtler, C., Hedlund, M., Fischer, H., Samuelsson, P., Svensson, M. and Svanborg, C. 2001. Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation. Mol. Microbiol. 40:37.[ISI][Medline]
  14. Perera, P. Y., Mayadas, T. N., Takeuchi, O., Akira, S., Zaks-Zilberman, M., Goyert, S. M. and Vogel, S. N. 2001. CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression. J. Immunol. 166:574.[Abstract/Free Full Text]
  15. Brightbill, H. D., Libraty, D. H., Krutzik, S. R., Yang, R. B., Belisle, J. T., Bleharski, J. R., Maitland, M., Norgard, M. V., Plevy, S. E., Smale, S. T., Brennan, P. J., Bloom, B. R., Godowski, P. J. and Modlin, R. L. 1999. Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. Science 285:732.[Abstract/Free Full Text]
  16. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K. and Akira, S. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11:443.[ISI][Medline]
  17. Underhill, D. M., Ozinsky, A., Hajjar, A. M., Stevens, A., Wilson, C. B., Bassetti, M. and Aderem, A. 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401:811.[ISI][Medline]
  18. Hou, L., Sasaki, H. and Stashenko, P. 2000. Toll-like receptor 4-deficient mice have reduced bone destruction following mixed anaerobic infection. Infect. Immun. 68:4681.[Abstract/Free Full Text]
  19. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K. and Akira, S. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740.[ISI][Medline]
  20. Tabeta, K., Yamazaki, K., Akashi, S., Miyake, K., Kumada, H., Umemoto, T. and Yoshie, H. 2000. Toll-like receptors confer responsiveness to lipopolysaccharide from Porphyromonas gingivalis in human gingival fibroblasts. Infect. Immun. 68:3731.[Abstract/Free Full Text]
  21. Campos, M. A., Almeida, I. C., Takeuchi, O., Akira, S., Valente, E. P., Procopio, D. O., Travassos, L. R., Smith, J. A., Golenbock, D. T. and Gazzinelli, R. T. 2001. Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite. J. Immunol. 167:416.[Abstract/Free Full Text]
  22. Okamura, Y., Watari, M., Jerud, E. S., Young, D. W., Ishizaka, S. T., Rose, J., Chow, J. C. and Strauss, J. F., 3rd. 2001. The extra domain A of fibronectin activates Toll-like receptor 4. J. Biol. Chem. 276:10229.[Abstract/Free Full Text]
  23. Seki, E., Tsutsui, H., Nakano, H., Tsuji, N., Hoshino, K., Adachi, O., Adachi, K., Futatsugi, S., Kuida, K., Takeuchi, O., Okamura, H., Fujimoto, J., Akira, S. and Nakanishi, K. 2001. Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1beta. J. Immunol. 166:2651.[Abstract/Free Full Text]
  24. Stassen, M., Muller, C., Arnold, M., Hultner, L., Klein-Hessling, S., Neudorfl, C., Reineke, T., Serfling, E. and Schmitt, E. 2001. IL-9 and IL-13 production by activated mast cells is strongly enhanced in the presence of lipopolysaccharide: NF-kappa B is decisively involved in the expression of IL-9. J. Immunol. 166:4391.[Abstract/Free Full Text]
  25. Takeuchi, O., Kawai, T., Muhlradt, P. F., Morr, M., Radolf, J. D., Zychlinsky, A., Takeda, K. and Akira, S. 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol. 13:933.
  26. Supajatura, V., Ushio, H., Nakao, A., Okumura, K., Ra, C. and Ogawa, H. 2001. Protective roles of mast cells against enterobacterial infection are mediated by toll-like receptor 4. J. Immunol. 167:2250.[Abstract/Free Full Text]
  27. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K. and Akira, S. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162:3749.[Abstract/Free Full Text]
  28. Hertz, C., Kiertscher, S., Godowski, P., Bouis, D., Norgard, M., Roth, M. and Modlin, R. 2001. Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J. Immunol. 166:2444.[Abstract/Free Full Text]
  29. Michelsen, K. S., Aicher, A., Mohaupt, M., Hartung, T., Dimmeler, S., Kirschning, C. J. and Schumann, R. R. 2001. The role of toll-like receptors (TLR) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2. J. Biol. Chem. 276:25680.[Abstract/Free Full Text]
  30. Takeuchi, O., Takeda, K., Hoshino, K., Adachi, O., Ogawa, T. and Akira, S. 2000. Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades. Int. Immunol. 12:113.[Abstract/Free Full Text]
  31. Ohashi, K., Burkart, V., Flohe, S. and Kolb, H. 2000. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 164:558.[Abstract/Free Full Text]
  32. Baumgarten, G., Knuefermann, P., Nozaki, N., Sivasubramanian, N., Mann, D. L. and Vallejo, J. G. 2001. In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4. J. Infect. Dis. 183:1617.[ISI][Medline]
  33. Jones, B. W., Means, T. K., Heldwein, K. A., Keen, M. A., Hill, P. J., Belisle, J. T. and Fenton, M. J. 2001. Different Toll-like receptor agonists induce distinct macrophage responses. J. Leukoc. Biol. 69:1036.[Abstract/Free Full Text]
  34. Kitamura, Y., Kakimura, J., Koike, H., Umeki, M., Gebicke-Haerter, P. J., Nomura, Y. and Taniguchi, T. 2001. Effects of 15-deoxy-delta(12,14) prostaglandin J(2) and interleukin-4 in Toll-like receptor-4-mutant glial cells. Eur. J. Pharmacol. 411:223.[ISI][Medline]
  35. Schroder, N. W., Opitz, B., Lamping, N., Michelsen, K. S., Zahringer, U., Gobel, U. B. and Schumann, R. R. 2000. Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids. J. Immunol. 165:2683.[Abstract/Free Full Text]
  36. Thoma-Uszynski, S., Stenger, S., Takeuchi, O., Ochoa, M. T., Engele, M., Sieling, P. A., Barnes, P. F., Rollinghoff, M., Bolcskei, P. L., Wagner, M., Akira, S., Norgard, M. V., Belisle, J. T., Godowski, P. J., Bloom, B. R. and Modlin, R. L. 2001. Induction of direct antimicrobial activity through mammalian toll-like receptors. Science 291:1544.[Abstract/Free Full Text]
  37. Tsan, M. F., Clark, R. N., Goyert, S. M. and White, J. E. 2001. Induction of TNF-alpha and MnSOD by endotoxin: role of membrane CD14 and Toll-like receptor-4. Am. J. Physiol. Cell Physiol. 280:C1422.
  38. Ozinsky, A., Underhill, D. M., Fontenot, J. D., Hajjar, A. M., Smith, K. D., Wilson, C. B., Schroeder, L. and Aderem, A. 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.[Abstract/Free Full Text]
  39. Hajjar, A. M., O’Mahony, D. S., Ozinsky, A., Underhill, D. M., Aderem, A., Klebanoff, S. J. and Wilson, C. B. 2001. Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J. Immunol. 166:15.[Abstract/Free Full Text]
  40. Kawai, T., Adachi, O., Ogawa, T., Takeda, K. and Akira, S. 1999. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115.[ISI][Medline]
  41. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389.[Abstract/Free Full Text]
  42. Lennon, G., Auffray, C., Polymeropoulos, M. and Soares, M. B. 1996. The IMAGE Consortium: an integrated molecular analysis of genomes and their expression. Genomics 33:151.[ISI][Medline]
  43. Kimura, S., Tamamura, T., Nakagawa, I., Koga, T., Fujiwara, T. and Hamada, S. 2000. CD14-dependent and independent pathways in lipopolysaccharide-induced activation of a murine B-cell line, CH12. LX. Scand. J. Immunol. 51:392.[ISI][Medline]
  44. Hardiman, G., Jenkins, N. A., Copeland, N. G., Gilbert, D. J., Garcia, D. K., Naylor, S. L., Kastelein, R. A. and Bazan, J. F. 1997. Genetic structure and chromosomal mapping of MyD88. Genomics 45:332.[ISI][Medline]
  45. Girolomoni, G., Lutz, M. B., Pastore, S., Assmann, C. U., Cavani, A. and Ricciardi-Castagnoli, P. 1995. Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin. Eur. J. Immunol. 25:2163.[ISI][Medline]
  46. Paglia, P., Girolomoni, G., Robbiati, F., Granucci, F. and Ricciardi-Castagnoli, P. 1993. Immortalized dendritic cell line fully competent in antigen presentation initiates primary T cell responses in vivo. J. Exp. Med. 178:1893.[Abstract]
  47. Granucci, F., Girolomoni, G., Lutz, M. B., Foti, M., Marconi, G., Gnocchi, P., Nolli, L. and Ricciardi-Castagnoli, P. 1994. Modulation of cytokine expression in mouse dendritic cell clones. Eur. J. Immunol. 24:2522.[ISI][Medline]
  48. Raschke, W. C., Baird, S., Ralph, P. and Nakoinz, I. 1978. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 15:261.[ISI][Medline]
  49. Ralph, P., Prichard, J. and Cohn, M. 1975. Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity. J. Immunol. 114:898.[Abstract]
  50. Ralph, P. and Nakoinz, I. 1975. Phagocytosis and cytolysis by a macrophage tumour and its cloned cell line. Nature 257:393.[ISI][Medline]
  51. Snyderman, R., Pike, M. C., Fischer, D. G. and Koren, H. S. 1977. Biologic and biochemical activities of continuous macrophage cell lines P388D1 and J774.1. J. Immunol. 119:2060.[ISI][Medline]
  52. MacKay, K., Striker, L. J., Elliot, S., Pinkert, C. A., Brinster, R. L. and Striker, G. E. 1988. Glomerular epithelial, mesangial, and endothelial cell lines from transgenic mice. Kidney Int. 33:677.[ISI][Medline]
  53. Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R. and Bistoni, F. 1990. Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J. Neuroimmunol. 27:229.[ISI][Medline]
  54. Arora, N., Min, K. U., Costa, J. J., Rhim, J. S. and Metcalfe, D. D. 1993. Immortalization of mouse bone marrow-derived mast cells with Ad12-SV40 virus. Int. Arch. Allergy Immunol. 100:319.[ISI][Medline]
  55. Dastych, J. and Metcalfe, D. D. 1994. Stem cell factor induces mast cell adhesion to fibronectin. J. Immunol. 152:213.[Abstract/Free Full Text]
  56. Koyasu, S., Nakauchi, H., Kitamura, K., Yonehara, S., Okumura, K., Tada, T. and Yahara, I. 1985. Production of interleukin 3 and gamma-interferon by an antigen-specific mouse suppressor T cell clone. J. Immunol. 134:3130.[Abstract/Free Full Text]
  57. Lanier, L. L. and Warner, N. L. 1981. Cell cycle related heterogeneity of Ia antigen expression on a murine B lymphoma cell line: analysis by flow cytometry. J. Immunol. 126:626.[Abstract/Free Full Text]
  58. Paige, C. J., Kincade, P. W. and Ralph, P. 1978. Murine B cell leukemia line with inducible surface immunoglobulin expression. J. Immunol. 121:641.[ISI][Medline]
  59. Weigert, M. G., Cesari, I. M., Yonkovich, S. J. and Cohn, M. 1970. Variability in the lambda light chain sequences of mouse antibody. Nature 228:1045.[ISI][Medline]
  60. Schubert, D., Jobe, A. and Cohn, M. 1968. Mouse myelomas producing precipitating antibody to nucleic acid bases and/or nitrophenyl derivatives. Nature 220:882.[ISI][Medline]
  61. Ljunggren, H. G. and Karre, K. 1985. Host resistance directed selectively against H-2-deficient lymphoma variants. Analysis of the mechanism. J. Exp. Med. 162:1745.[Abstract]
  62. Haran-Ghera, N., Ben-Yaakov, M. and Peled, A. 1977. Immunologic characteristics in relation to high and low leukemogenic activity of radiation leukemia virus variants. I. Cellular analysis of immunosuppression. J. Immunol. 118:600.[Abstract]
  63. Gillis, S. and Smith, K. A. 1977. Long term culture of tumour-specific cytotoxic T cells. Nature 268:154.[ISI][Medline]
  64. Greenberger, J. S., Sakakeeny, M. A., Humphries, R. K., Eaves, C. J. and Eckner, R. J. 1983. Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hemato poietic progenitor cell lines. Proc. Natl Acad. Sci. USA 80:2931.[Abstract]
  65. Valtieri, M., Tweardy, D. J., Caracciolo, D., Johnson, K., Mavilio, F., Altmann, S., Santoli, D. and Rovera, G. 1987. Cytokine-dependent granulocytic differentiation. Regulation of proliferative and differentiative responses in a murine progenitor cell line. J. Immunol. 138:3829.[Abstract/Free Full Text]
  66. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S. and Steinman, R. M. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract]
  67. Sebastiani, G., Leveque, G., Lariviere, L., Laroche, L., Skamene, E., Gros, P. and Malo, D. 2000. Cloning and characterization of the murine toll-like receptor 5 (Tlr5) gene: sequence and mRNA expression studies in Salmonella-susceptible MOLF/Ei mice. Genomics 64:230.[ISI][Medline]
  68. Du, X., Poltorak, A., Wei, Y. and Beutler, B. 2000. Three novel mammalian toll-like receptors: gene structure, expression, and evolution. Eur. Cytokine Netw. 11:362.[ISI][Medline]
  69. Underhill, D. M., Ozinsky, A., Smith, K. D. and Aderem, A. 1999. Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages. Proc. Natl Acad. Sci. USA 96:14459.[Abstract/Free Full Text]
  70. Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N. and Weis, J. J. 2000. Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2. J. Immunol. 165:618.[Abstract/Free Full Text]
  71. Qureshi, S. T., Lariviere, L., Leveque, G., Clermont, S., Moore, K. J., Gros, P. and Malo, D. 1999. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J. Exp. Med. 189:615.[Abstract/Free Full Text]
  72. Xu, Y., Tao, X., Shen, B., Horng, T., Medzhitov, R., Manley, J. L. and Tong, L. 2000. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 408:111.[ISI][Medline]
  73. Horng, T., Barton, G. M. and Medzhitov, R. 2001. TIRAP: an adapter molecule in the Toll signaling pathway. Nat. Immunol. 2:835.[ISI][Medline]
  74. Alexopoulou, L., Holt, A. C., Medzhitov, R. and Flavell, R. A. 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll- like receptor 3. Nature 413:732.[ISI][Medline]
  75. Visintin, A., Mazzoni, A., Spitzer, J. H., Wyllie, D. H., Dower, S. K. and Segal, D. M. 2001. Regulation of Toll-like receptors in human monocytes and dendritic cells. J. Immunol. 166:249.[Abstract/Free Full Text]
  76. Muzio, M., Bosisio, D., Polentarutti, N., D’Amico, G., Stoppacciaro, A., Mancinelli, R., van’t Veer, C., Penton-Rol, G., Ruco, L. P., Allavena, P. and Mantovani, A. 2000. Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J. Immunol. 164:5998.[Abstract/Free Full Text]
  77. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D. and Modlin, R. L. 2000. Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10. J. Immunol. 165:3804.[Abstract/Free Full Text]
  78. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B. and Palucka, K. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767.[ISI][Medline]
  79. Malaviya, R., Ikeda, T., Ross, E. and Abraham, S. N. 1996. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature 381:77.[ISI][Medline]
  80. Echtenacher, B., Mannel, D. N. and Hultner, L. 1996. Critical protective role of mast cells in a model of acute septic peritonitis. Nature 381:75.[ISI][Medline]
  81. Church, M. K., Norn, S., Pao, G. J. and Holgate, S. T. 1987. Non-IgE-dependent bacteria-induced histamine release from human lung and tonsillar mast cells. Clin. Allergy 17:341.[ISI][Medline]
  82. Abraham, S. N. and Malaviya, R. 1997. Mast cells in infection and immunity. Infect. Immun. 65:3501.[Free Full Text]
  83. Leal-Berumen, I., Conlon, P. and Marshall, J. S. 1994. IL-6 production by rat peritoneal mast cells is not necessarily preceded by histamine release and can be induced by bacterial lipopolysaccharide. J. Immunol. 152:5468.[Abstract/Free Full Text]
  84. Smith, J. A. 1994. Neutrophils, host defense, and inflammation: a double-edged sword. J. Leukoc. Biol. 56:672.[Abstract]
  85. Scapini, P., Lapinet-Vera, J. A., Gasperini, S., Calzetti, F., Bazzoni, F. and Cassatella, M. A. 2000. The neutrophil as a cellular source of chemokines. Immunol. Rev. 177:195.[ISI][Medline]
  86. Surette, M. E., Palmantier, R., Gosselin, J. and Borgeat, P. 1993. Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen. J. Exp. Med. 178:1347.[Abstract]
  87. Aida, Y. and Pabst, M. J. 1990. Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. Requirement for plasma but not for tumor necrosis factor-alpha. J. Immunol. 145:3017.[Abstract/Free Full Text]
  88. Jakway, J. P., Usinger, W. R., Gold, M. R., Mishell, R. I. and DeFranco, A. L. 1986. Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide, and other bacterial products. J. Immunol. 137:2225.[Abstract/Free Full Text]
  89. Krieg, A. M., Yi, A. K., Matson, S., Waldschmidt, T. J., Bishop, G. A., Teasdale, R., Koretzky, G. A. and Klinman, D. M. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546.[ISI][Medline]
  90. Liang, H., Reich, C. F., Pisetsky, D. S. and Lipsky, P. E. 2000. The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides. J. Immunol. 165:1438.[Abstract/Free Full Text]
  91. Arias, M. A., Rey Nores, J. E., Vita, N., Stelter, F., Borysiewicz, L. K., Ferrara, P. and Labeta, M. O. 2000. Cutting edge: human B cell function is regulated by interaction with soluble CD14: opposite effects on IgG1 and IgE production. J. Immunol. 164:3480.[Abstract/Free Full Text]
  92. Triantafilou, K., Triantafilou, M. and Dedrick, R. L. 2001. A CD14-independent LPS receptor cluster. Nat. Immunol. 2:338.[ISI][Medline]
  93. Gery, I., Kruger, J. and Spiesel, S. Z. 1972. Stimulation of B-lymphocytes by endotoxin. Reactions of thymus-deprived mice and karyotypic analysis of dividing cells in mice bearing T 6 T 6 thymus grafts. J. Immunol. 108:1088.[ISI][Medline]
  94. Andersson, J., Moller, G. and Sjoberg, O. 1972. Selective induction of DNA synthesis in T and B lymphocytes. Cell. Immunol. 4:381.[ISI][Medline]
  95. Matsuguchi, T., Musikacharoen, T., Ogawa, T. and Yoshikai, Y. 2000. Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages. J. Immunol. 165:5767.[Abstract/Free Full Text]
  96. Mokuno, Y., Matsuguchi, T., Takano, M., Nishimura, H., Washizu, J., Ogawa, T., Takeuchi, O., Akira, S., Nimura, Y. and Yoshikai, Y. 2000. Expression of toll-like receptor 2 on gamma delta T cells bearing invariant V gamma 6/V delta 1 induced by Escherichia coli infection in mice. J. Immunol. 165:931.[Abstract/Free Full Text]