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Department of Molecular Biology, Genentech Inc., South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of most TLRs leads to recruitment of MyD88, a homolog of
Drosophila Tube that contains an intracellular signaling
domain and a death domain (28). MyD88 interacts with IL-1
receptor-associated kinase, leading to initiation of a signal
transduction cascade culminating in nuclear translocation of NF-
B
family members and altered gene expression. In addition to
MyD88-mediated signaling, TLR2 has been shown to interact with the Rac1
GTPase, thereby initiating a signaling cascade that also results in
NF-B translocation and possibly other nontranscriptional cellular
responses (29). Chimeras with intracellular signaling
domains of TLRs coupled to the extracellular domain of CD4 demonstrate
that many TLRs can cause NF-B transcriptional activation (1, 4).
To explore settings in which human TLRs may play a role, we surveyed the expression of all known TLR mRNAs in normal human tissues and in cells activated by microbial or inflammatory compounds. The results suggest that dynamic regulation of TLR expression may be an important consideration in the elucidation of TLR biology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The monocytic leukemia cell line THP-1 was grown in RPMI 1640 with 10% FCS, 10 mM HEPES, 2 mM glutamine, penicillin, and streptomycin (100 U/ml each) at 37°C in a 5% CO2 atmosphere. Escherichia coli (ATCC 4157) and S. aureus (ATCC 12598) were stored at -80°C as DMSO stocks and grown in Luria broth. Log phase subcultures were grown at 37°C, washed in PBS, and quantitated turbidometrically.
Stimuli
THP-1 cells were differentiated with 10 ng PMA/ml for 12–18 h.
LPS was purchased from List Biological Laboratories (Campbell, CA) and
re-extracted to remove contaminating endotoxin proteins as described
(30). Stimulation of cells was performed in growth medium
supplemented with 10% FCS as a source for LPS-binding protein.
Synthetic bacterial lipopeptide (sBLP;
N-
-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-L-cysteine-L-serine-L-lysine4),
was a gift from A. Zychlinsky (New York University School of Medicine,
New York, NY). Cytokines were purchased from Calbiochem (La Jolla, CA)
or Oncogene Research Products (San Diego, CA).
Taqman
Commercially available cDNA pools (Clontech Laboratories, Palo
Alto, CA) were prepared by the manufacturer from DNase-treated
poly(A)-selected RNA purified from tissues of healthy adults (Table I
). PCR using primers designed to amplify
a 943-bp TNF-
product from genomic templates and a 610-bp TNF-
product from cDNA did not detect any 943-bp products after 40 cycles.
Pools of cDNA were normalized simultaneously to four separate
housekeeping genes (-tubulin, -actin, G3PDH, and
phospholipase A2) by the manufacturer. RNA used
in
Figs. 3–5 were prepared from cells using a Qiagen (Valencia, CA)
Mini or Midi RNA-easy kit and random-primed cDNA prepared using the
Clontech reverse transcription kit as per manufacturer’s instructions.
DNase treatment was included for isolation of RNA from primary cells.
Taqman probe/primer combinations used in these studies were identified
using the Primer Express software package (Applied Biosystems, Foster
City, CA) and are presented in Table II.
All primers were stored at a concentration of 10 OD/ml, and 0.05
µl of each forward and reverse primer as well as 0.1 µl of
the probe were used per 25-µl reaction. Taqman PCR was performed in
25-µl volumes using AmpliTaq Gold polymerase and universal
reaction buffer with 5.5 mM MgCl2 (Applied
Biosystems). Threshold cycle numbers (Ct) were determined
with Sequence Detector Software (version 1.6; Applied Biosystems) and
transformed using the 
Ct or Ct methods
as described by the manufacturer using GAPDH or
RPL19 as the calibrator genes where indicated.
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Human venous blood was obtained after informed consent and was anticoagulated with citrate. Blood was placed in sterile 50-ml polypropylene tubes and incubated for 6 h either with buffer alone or 1 x 105 E. coli/ml at 37°C in a humidified 5% CO2 atmosphere with intermittent mixing. Blood was then mixed with 2 volumes of ice cold HBSS(-), overlaid on Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ) and centrifuged for 40 min at 400 x g at 20°C. Mononuclear cells were aspirated from the interface and washed in HBSS(-) supplemented with 0.5% human serum albumin. The RBC/granulocyte phase was subjected to isotonic RBC lysis using 10 volumes of 123 mM NH4Cl, 8 mM KHCO3, and 25 µM EDTA. After centrifugation, granulocytes were resuspended in lysis buffer followed with 4 volumes of HBSS(-) for a final wash. Washed mononuclear cells were further subjected to positive selection using anti-CD14-conjugated paramagnetic beads and purified using the AutoMACs system per manufacturer’s instructions (Miltenyi Biotec, Auburn, CA).
Immunoprecipitation of TLRs from THP1 cells
THP-1 cells were treated as described above and then extracted in lysis buffer (1% Triton X-100, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 25 mM HEPES, pH 7.2) supplemented with 50 µg/ml PMSF for 1 h at 4°C, and then centrifuged for 10 min at top speed in an Eppendorf microfuge (Beckman Coulter, Fullerton, CA). Supernatants were incubated rotating at 4°C with 5 µg mouse mAbs against human TLR1 (mAb 2736) or TLR4 (mAb 2723) per 500 µl detergent extract with 25 µl protein A/G beads (Pierce, Rockford, IL). Beads were washed three times with lysis buffer and then boiled in 6x SDS-PAGE sample buffer, and the eluted proteins were resolved on a Novex 4–12% SDS-PAGE gel and silver stained using the SilverSNAP kit (Pierce).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Distribution of human TLR mRNA in adult tissues was assayed by
Taqman, a real-time fluorescence PCR technique that provides sensitive
detection and quantitative enumeration of sequences over at least 7
orders of magnitude of starting number. Human tissue cDNA pools were
prenormalized by the vendor (Clontech) to four housekeeping genes and
shown to be devoid of contaminating genomic DNA as described in
Materials and Methods. To facilitate comparison between
tissues, all samples were normalized to the expression of the
particular TLR in spleen (spleen = 1). As shown in Fig. 1, tissues involved in immune function
(e.g., spleen, peripheral blood leukocytes) displayed the most diverse
repertoire of TLR mRNAs, as did those expected to encounter microbes
(e.g., lung, small intestine, colon). TLR3 and TLR5 were expressed
ubiquitously, whereas RP105 and TLR10 were more restricted.
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Because peripheral blood cells expressed high levels of TLR
mRNAs compared with other tissues, we next examined TLR
expression in purified subpopulations containing >95% pure
CD4+, CD8+,
CD14+, or CD19+ leukocytes.
The cDNAs were prenormalized as above, and the data were normalized to
the expression of the target sequence in the
CD14+ population. Although expression of MyD88
was similar in all cell types (±2.5-fold of the average), relative
expression of specific TLR mRNAs varied up to 3 orders of magnitude
(Fig. 2). Several TLRs (e.g., TLR9,
TLR10, and RP105) were expressed at highest levels in
CD19+ B cells,whereas others (e.g., TLR2,
TLR4, and TLR8) were expressed maximally in CD14+
mononuclear cells. These findings have been reproduced independently
using RNA prepared from leukocytes purified by magnetic immune
selection (data not shown.)
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Because CD14+ cells expressed the widest variety of TLRs at high levels and are thought to play primary roles in the initial phases of the innate immune response, we initially focused on identifying agents that alter the expression of TLR mRNA in the monocyte-like leukemic THP-1 cell line.
Differentiation of THP-1 by treatment with PMA increases their
sensitivity to LPS by several orders of magnitude as measured in an
assay of TNF production (data not shown). To determine whether enhanced
sensitivity to LPS could be due to changes in TLR expression, we
analyzed by Taqman the expression of TLRs and associated molecules in
undifferentiated and PMA-treated THP-1 cells (Fig. 3
A). In addition to
significant up-regulation of the LPS receptor TLR4, RP105 was also
significantly up-regulated in these cells as were the mRNAs for CD14,
MD2, and MyD88 and other TLRs not known to be involved in LPS signaling
(e.g., TLR1, TLR6, TLR7, TLR8). Treatment of THP-1 cells with LPS
altered expression of several TLR mRNAs in both the presence and the
absence of PMA differentiation. For example, LPS treatment increased
mRNA levels of TLR 1–8 but not TLR9 or TLR10 mRNAs. Interestingly, the
90-fold PMA-induced increase in RP105 expression was abolished by
treatment with LPS. MyD88 mRNA was up-regulated by LPS and by PMA,
presumably to accommodate the increased amounts of TLR proteins
synthesized in these cells. Fig. 3B demonstrates that
for TLR1 and TLR4, protein expression correlates with mRNA expression
shown in Fig. 3A.
The effects of both PMA and LPS on regulation of TLR message levels may
be due either to direct effects or to autocrine stimulation by secreted
factors such as cytokines. To test this possibility, we treated
PMA-differentiated THP-1 cells with several different cytokines and
performed Taqman analysis after 6 h of treatment. As shown in Fig. 3C, all agents tested had measurable effects on the
expression of TLR mRNAs. TLR8 expression, although moderately
responsive to all stimuli tested, increased >40-fold with IFN-
treatment. IL-6, which also increased expression of all TLRs, increased
TLR7 expression >12-fold. Thus, a variety of cytokines induced by
microbial products such as LPS may also alter TLR expression.
Remarkably, specific agonists of TLR2 and TLR4 (sBLP and LPS,
respectively) differentially altered the expression of other TLRs. For
example, whereas TLR3 is up-regulated by LPS, it is repressed by sBLP.
Also, whereas bacterial lipopeptide down-regulated RP105 by 2-fold, LPS
down-regulated RP105 mRNA by 100-fold, suggesting that the consequences
of activation of different TLRs are distinct.
To identify changes in TLR expression in response to a more
physiological stimulus, we coincubated PMA-differentiated THP-1 cells
with live bacteria. As shown in Fig. 4, both Gram-positive (S. aureus) and Gram-negative (E.
coli) bacteria altered expression of several TLRs. Although TLR2,
-5, -7, -8, and -10 appeared to be similarly affected, expression of
TLR3 was more responsive to Gram-positive bacteria (7-fold) than to
Gram-negative bacteria (
2-fold). RP105 was down-regulated >30-fold
by E. coli, whereas S. aureus caused <10-fold
down-regulation.
Changes in TLR expression levels in response to microbial challenge of primary leukocytes
Innate immune responses in vivo are orchestrated through the
activation of a variety of cellular and humoral systems that may
haveboth positive and negative effects on cellular activation.
To test whether the changes seen in TLR expression in simple in vitro
settings were mirrored in more complex ex vivo settings, human venous
blood was anticoagulated with citrate and incubated with 1 x
105 E. coli per ml for 6 h. RNA
was isolated from monocytes and granulocytes and analyzed for TLR
expression. As shown in Fig. 5A, mRNA expression levels for
TLR2, TLR3, and MyD88 were similar whereas granulocytes express more
TLR1, -4, -6, and -8–10. In contrast, monocytes express more TLR5 and
almost 60 times more TLR7 than granulocytes. The expression of RP105
and its chaperone MD1 is >100-fold higher in monocytes than in
granulocytes, whereas expression of TLR4 and its chaperone
MD2 are 5- to 10-fold lower in monocytes than in granulocytes.
Treatment of whole blood with E. coli resulted in significant changes in the expression of several TLRs and associated molecules. TLR1, TLR6, and TLR9 were down-regulated in both cell types, whereas TLR3, TLR7 and MyD88 were up-regulated. Both TLR8 and TLR10 were up-regulated in monocytes but down-regulated in granulocytes. Microbe-induced differential regulation of TLR mRNA by phagocytes in whole blood suggests active modification of cellular responsiveness of these cells during the course of infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with their roles in immune surveillance, TLR mRNAs
are expressed at higher levels in tissues exposed to the external
environment such as lung and the gastrointestinal tract as well as in
immunologically important settings such as peripheral blood leukocytes
and spleen (Fig. 1). TLR3 and TLR5, which share significant sequence
homology, are both expressed broadly at levels comparable with those in
spleen (perhaps suggesting a general role in different tissues) and are
both regulated by bacteria and their products (Figs. 4 and 5). Although
TLR5 has been shown to mediate a signal in response to bacterial
flagellin (23, 24), no function has yet been associated
with TLR3. Human intestinal epithelial cells constitutively express
TLR3 and decrease its expression in Crohn’s disease but not in
ulcerative colitis (35). The demonstration that TLR3 is
expressed in epithelia and in monocytes, granulocytes, T cells, and B
cells differs from a report by Muzio et al. (36) who
detected expression only in monocyte-derived ex vivo-differentiated
dendritic cells.
The pattern of TLR expression in different peripheral blood leukocyte populations implies specific roles in each population. Although CD14+ cells expressed less TLR3, TLR9, and TLR10 than other cells, they expressed the highest levels of TLR2, TLR4, TLR5, and TLR8, and levels of TLR1, TLR6, and TLR7 were comparable to those of CD19+ cells. Similar to RP105, which was previously reported to be a B cell surface protein (37), TLR9 and TLR10 are expressed in B cells at levels at least 50 times greater than in other blood cells. TLR1, which has been reported to enhance TLR2 signaling in response to some agonists (27) and inhibit responses to others (26), was expressed at roughly similar levels in all leukocyte populations tested and also in lung and pancreas. MyD88 expression was also similar in all isolated cell populations tested and was detectable throughout the body including in nonmyeloid tissues. Although MyD88 also serves as a signaling intermediate in other pathways (e.g., IL-1R), its broad expression is suggestive of general importance of TLR function in most cells. Although mRNA expression data cannot exclude the possibility that small amounts of TLRs expression seen in some cells types may be functionally important, other data suggest that TLR mRNA levels correlate with their function, i.e., cell lines expressing relatively little TLR4 as detected by Taqman (e.g., HEK 293, not shown) are hyporesponsive to LPS (38). However, because TLR mRNAs are strongly responsive to a variety of stimuli (see below), cellular responsiveness to a given TLR agonist may differ drastically on prior activation by other TLR agonists.
To characterize determinants of TLR mRNA expression in a
well-controlled in vitro setting, we selected the THP-1 acute monocytic
leukemia cell line for our studies. In the presence of PMA, these cells
differentiate toward a more monocyte-like phenotype (39)
and coordinately up-regulate many TLRs (Fig. 3). For example, PMA
increases TLR1 mRNA expression 50-fold, whereas TLR-4, -6, -7, and -8
expression increased between 5- and 7-fold. The TLR chaperones MD1 and
MD2 were also PMA responsive, with MD-2 mRNA increasing at least
90-fold. In addition to being required for TLR4 LPS responsiveness
(13), MD2 also increases TLR2 responsiveness to
TLR2-specific agonists (40). Strikingly, the expression of
RP105, which was reported to be a B cell marker (37) and
to mediate LPS-signaling in B cells (19), increased
>70-fold during differentiation, leading us to hypothesize that RP105
is also important in monocyte function. Both MyD88 and CD14 expression
increased significantly and with increased expression of TLR4 and
possibly RP105, likely contribute to the strong increase in sensitivity
of THP-1 cells to LPS during maturation. Endotoxin protein-depleted LPS
prepared as described (30) further increased the
expression of many TLR mRNAs. TLR8, which was up-regulated 5-fold in
response to PMA alone, was further up-regulated by LPS to a final
expression level 50 times that of untreated undifferentiated THP-1
cells. RP105 was strongly suppressed on LPS stimulation (100-fold)
perhaps contributing to the LPS-induced hyposensitivity of monocytic
cells to secondary LPS stimulation.
A hallmark of cellular responses to activation of innate immune systems
is the release of cytokines such as TNF-, IL-1, and IFN-.
Interestingly, several TLR mRNAs were specifically up-regulated by
particular cytokines in PMA-differentiated THP-1 cells. For example,
IL-6 increased TLR7 expression by >10-fold, whereas IFN- increased
TLR8 expression by >40-fold. Thus, differential production of
cytokines in particular pathological contexts may impact the spectrum
of TLR-mediated responses of which cells are capable in that setting.
Although up-regulation of MyD88 by IL-6 was previously reported
(41), a variety of stimuli (e.g., PMA, LPS) increased its
expression possibly to accommodate the increased expression of TLRs.
Several studies found differential regulation of TLR2 and TLR4 in
response to microbial products and some cytokines
(42, 43, 44, 45). Interestingly, although activation of mouse
monocytes with LPS causes little change in the total level of TLR4
mRNA, the abundance of a splice variant mRNA encoding a secreted
antagonist form is strongly increased (46).
Treatment of PMA-differentiated THP-1 cells with specific agonists of TLR2 (sBLP) or of TLR4 (endotoxin protein-depleted LPS) caused different changes in the expression of other TLR mRNAs. Although both up-regulated TLR8 to a similar extent, TLR3 was up-regulated 3-fold by LPS and repressed 2.5-fold by BLP. RP105 was down-regulated 100-fold in response to LPS but only 2-fold by BLP. Thus, in humans, like Drosophila, activation of different PAMP receptors specifically alters host transcriptional responses presumably to better contend with the particular activating pathogen. Flies defective in Toll signaling are unable to up-regulate antifungal cytotoxic peptides and succumb to fungal infection (47), whereas flies with defects in another TLR family member, termed 18-wheeler, are unable to appropriately sense bacterial pathogens and fail to produce antibacterial peptides (48). The simplest interpretation of the observations in THP-1 cells is that activation of different mammalian TLRs likewise results in specific transcription factor activation and responses. A recent study has also demonstrated some differences in gene expression due to activation by selective TLR2 and TLR4 agonists (49). Interestingly, a likely agonist of TLR9, Shigella plasmid DNA, causes a down-regulation of human antimicrobial peptides in monocytes (50). Furthermore, activation of TLR2 by bacterial lipoproteins leads to apoptosis in a variety of host cells, notably monocytes (7, 51). Thus, some microbes may use TLR receptors to down-regulate innate immune systems during infection.
In vitro experiments with single cell types in simple laboratory medium
rarely replicate the complex networks of interactions between humoral
and cellular agents active in settings like human blood. We therefore
examined steady state levels of TLR mRNA in different cell populations
after incubation in human blood ex vivo with E. coli.
Relative to unstimulated monocytes, granulocytes express higher levels
of most TLR mRNAs with the exception of TLR7, which is expressed
60-fold higher in monocytes than in granulocytes. We also found that
granulocytes express >100-fold less RP105 and MD1 than monocytes,
again suggesting that these proteins play a role in monocyte function.
Similar to results seen in vitro with THP-1 cells, primary monocytes
also down-regulate their RP105 in response to E. coli.
Interestingly, two TLRs with significant down-regulation in response to
E. coli are TLR1 and TLR6, both of which have been shown to
alter the ligand specificity of other TLRs (26, 27).
Although classically considered to be terminally differentiated,
granulocytes express several NF-B/Rel proteins that undergo nuclear
translocation on activation (52), leading to cellular
changes including alterations in the expression of TLRs in response to
bacterial stimulation. The expression of TLRs in granulocytes, the most
abundant professional phagocyte, underscores further the essential
biological role of the TLRs in first line innate immune defenses
in vivo.
Note Added in Proof.
L. Alexopoulou et al. (53) recently demonstrated that TLR3 is a receptor for viral double-stranded RNA.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: PAMP, pathogen-associated microbial product; TLR, Toll-like receptor; BLP, bacterial lipopeptide; sBLP, synthetic bacterial lipopeptide.
Received for publication June 7, 2001. Accepted for publication November 7, 2001.
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B and expression of the inducible cyclooxygenase. J. Biol. Chem. 275:34035.B activation requires a Rac1-dependent pathway. Nat. Immunol. 1:533.[Medline]
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