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*
Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892-1360; and
Division of Molecular and Genetic Medicine, University of Sheffield, Sheffield, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
expression of most TLRs transiently increased and then nearly
disappeared. Stimulation of iDC, but not mDC, with LPS resulted in the
activation of IL-1 receptor-associated kinase, an early component in
the TLR signaling pathway, strongly suggesting that LPS signals through
a TLR. Surface expression of TLRs 1 and 4, as measured by mAb binding,
was very low, corresponding to a few thousand molecules per cell in
monocytes, and a few hundred or less in iDC. We conclude that TLRs are
expressed in iDC and are involved in responses to at least one
pathogen-derived substance, LPS. If TLR4 is solely responsible for LPS
signaling in humans, as it is in mice, then its extremely low surface
expression implies that it is a very efficient signal transducer in
iDC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (6) and results in the
up-regulation of HLA-DR, CD83, B7-1, B7-2, and CD40, and the production
of cytokines such as IL-12 and TNF-. By inducing maturation,
receptors for LPS and other pathogen-associated molecular patterns
(PAMPs) on iDC play pivotal roles in the development of adaptive
responses to Ags.
The innate recognition of PAMPs is mediated by genomically encoded
pattern recognition receptors (7, 8). Recently, the
Drosophila receptor, Toll, was shown to be essential for
protective immunity to fungal infections in flies (9), and
since then a number of immunologically relevant homologs of Toll have
been discovered in organisms as disparate as plants, insects, and
mammals (8, 10, 11, 12, 13). In humans, six Toll-like receptor
(TLR) homologs have been published to date (14, 15, 16, 17, 18), and
at least four others have been identified (12). All are
type I integral membrane receptors with extracellular leucine rich
regions and intracellular portions that are homologous to the signaling
domain of the IL-1R. The extracellular domain of human TLR4 associates
with a second protein, MD2, which is required for optimal LPS-induced
signaling (19). In transfection experiments, human TLRs 2
and 4 recruit and activate IL-1 receptor-associated kinase (IRAK) in
response to a variety of PAMPs, including the Gram-negative bacterial
toxin, LPS, resulting in downstream activation of NFB and c-Jun
NH2-terminal kinase, and secretion of IL-8 and
IL-12 (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). In mice, gene knockout studies indicate
that TLR4, but not TLR2, is required for LPS responsiveness, whereas
TLR2 is essential for responses to several Gram-positive PAMPs
(33, 34). The functions of other TLRs have yet to be
defined.
To understand the roles TLRs play in mammalian immunity, it is
essential to define their expression patterns in normal cells and
tissues. This is particularly important in DC, where PAMP recognition
drives their maturation. Therefore, we asked whether DC express TLRs,
and how TLR expression patterns change as monocytes differentiate into
iDC and mature into mDC. In this report we followed mRNA expression for
TLRs 1–5 and MD2 by Northern analysis and compared these results with
surface expression using mAbs against TLRs 1 and 4. We observed a
characteristic pattern of TLR and MD2 expression at each stage of DC
differentiation, and found that LPS induced striking, cell-specific
changes in the expression of TLRs in both iDC and monocytes. Moreover,
we demonstrate that LPS induces IRAK activation and TNF- secretion
in iDC, which express most TLRs, but not in mDC, which lack TLR
expression, suggesting that TLRs play an important role in iDC
activation and maturation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immature human DC were generated according to Sallusto et al.
(5). Briefly, elutriated monocytes from healthy National
Institutes of Health Blood Bank donors were cultured in DC medium
(complete medium (RPMI 1640 containing 10% heat-inactivated FCS, 2 mM
glutamine, 100 U/ml penicillin, and 55 µM 2-ME) supplemented with 50
ng/ml recombinant human GM-CSF and 34.5 ng/ml recombinant human IL-4
(PeproTech, Rocky Hill, NJ)), for 6–8 days. To induce maturation, iDC
were treated in DC medium with 100 ng/ml LPS (Escherichia
coli serotype 026:B6; Sigma, St. Louis, MO) unless stated
otherwise, in which case they were treated with 20 ng/ml recombinant
human TNF- (PeproTech) for the indicated periods of time.
Flow cytometry
Cell surface staining was performed using the following
anti-human mAbs from PharMingen (San Diego, CA):
anti-CD3PE;
anti-CD14PE;
anti-CD19FITC;
anti-CD80FITC;
anti-CD86FITC;
anti-HLA-DRFITC;
anti-CD1aPE; and
anti-CD83PE. The FITC- labeled anti-CD64
mAb (32.2) was a gift from Dr. Michael Fanger (Dartmouth Medical
School, Hanover, NH). The GD2.F4 mAb (mouse IgG1) was raised against
the extracellular domain of human TLR1 and was shown to be specific for
TLR1 in transfection experiments (43). The mAb against
TLR4, HTA1216 (mouse IgG1), has been described previously
(35) and was a gift of Dr. Kensuke Miyake (Saga Medical
School, Saga, Japan). The binding of both anti-TLR mAbs was
detected using a FITC-labeled goat anti-mouse secondary reagent
(Boehringer Mannheim, Indianapolis, IN) and was compared with cells
treated with an isotype-matched nonbinding control mAb (MOPC 300) and
with the mIgG1 anti-CD44 mAb, NIH44.1 (36). The
NIH44.1 mAb was also radioiodinated and used to determine the number of
molecules bound per cell at saturation (37). Staining was
performed in the presence of 100 µg/ml nonimmune human IgG to block
nonspecific binding to Fc
R. Ten thousand cells were acquired for
each sample, and dead cells were gated out based on their light scatter
properties. DC preparations always contained <5% lymphocytes based on
light scatter and staining for B and T cell markers. Cells from the
same donor were used when comparisons were made between monocytes, iDC,
and mDC.
Mixed lymphocyte reaction
DC were washed, irradiated (30 Gy), and added as stimulator cells to 96-well plates containing 1 x 105 responder cells per well. Responder cells were allogeneic T cells (>95% CD3+) purified from PBMC by negative selection using a mixture of mouse anti-human-CD14, -CD19, and -CD16 Abs (PharMingen) and sheep anti-mouse IgG-coated Dynabeads (Dynal, Oslo, Norway). After a 3-day stimulation, cells were pulsed with 5 µCi/ml of [3H]thymidine (NEN, Boston, MA) for 16 h, then harvested, and incorporation was measured by scintillation counting. Data are expressed as cpm (mean ± SD) of triplicate cultures.
Probes
Specific probes for TLRs 1–5 were generated by RT-PCR using
total RNA from PBL obtained from normal donors and the primer pairs
indicated in Table I
. Probe lengths and
sequences are also indicated in Table I. The 
-actin and GAPDH probes
were generated using primers from Promega (Madison, WI). Amplified
products were cloned into pCR-Blunt II-TOPO (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions, and the identity of each
insert was confirmed by automated DNA sequencing. Inserts were removed
by EcoRI digestion, gel purified, and random primed using
[-32P]dCTP. TLRs 1–5 are only distantly
related, and as expected, do not cross-hybridize as indicated by the
distinct size of each message seen in the Northern blots. However, TLRs
1 and 6 (accession number, AB020807) are highly homologous isoforms,
and our TLR1 probe would likely cross-hybridize with TLR6
(17). The size of the TLR6 message is unknown
(17), and if it is the same as TLR1, our TLR1 Northern
results could include contributions from the TLR6 isoform. The
full-length cDNAs of hMD2 and hCD14 were gifts from Dr. Kensuke Miyake
and Dr. Brian Seed (Massachusetts General Hospital, Boston, MA),
respectively.
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Total RNA from 
107 cells/point was
prepared using Trizol extraction (Life Technologies, Gaithersburg, MD).
Ten micrograms of total RNA from each sample was then analyzed using
standard Northern blotting procedures (hybridization was for at least
16 h at 42°C in 10 ml Hybrisol I (Intergen, Purchase, NY)
containing 25 ng probe). Each blot shown in this paper is
representative of data obtained from three separate donors. In some
cases membranes were probed twice; after hybridization with the first
probe, membranes were stripped by adding boiling 1% SDS and then
hybridized with the second probe. Equal loading was confirmed by
ethidium bromide staining of RNA in the original gel before transfer.
Where shown, each ethidium bromide gel represents one of several used
in data analysis. Transcript sizes and banding patterns correlated with
previously reported data (16, 38).
TNF- secretion
iDC or mDC (5 x 105; matured for
24 h in LPS and rested for 2 h in DC medium) were treated
with 100 ng/ml of E. coli LPS (055:B5; Sigma) or 50 ng/ml
PMA + 1 µg/ml ionomycin in 200 µl DC medium for 5 h. Duplicate
50-µl samples of supernatants were assayed for TNF- content using
a human TNF- ELISA kit (Endogen, Woburn, MA), which has a limit of
detection of 20 pg/ml.
IRAK assay
iDC or mDC (5 x 106 cells per point,
mDC were matured for 20 h in LPS and rested for 2 h in DC
medium) were stimulated with 1 µg/ml of LPS (45 min) or 10 ng/ml of
IL-1 (10 min). Where indicated, cells were preincubated for 10 min
with 5 µg of an anti IL-1 mAb (R&D Systems, Minneapolis, MN).
Cells were then lysed in 1 ml of lysis buffer (0.4% Nonidet P-40, 60
nM n-octyl--D-glucopyranoside, 137
mM NaCl, 2 mM EDTA, 50 mM NaF, 10% glycerol, 1 mM PMSF, 1 mM sodium
orthovanadate, and 10 µg/ml each of leupeptin and aprotinin (Sigma)).
Lysates were centrifuged for 10 min at 12,000 x g,
4°C and IRAK was immunoprecipitated for 12 h at 4°C from 700
µl of supernatant using 20 µl of packed protein A-Sepharose
(Amersham Pharmacia Biotech, Piscataway, NJ) coupled with 2 µg of
anti IRAK pAb (Upstate Biotechnology, Waltham, MA). The beads were
washed three times in lysis buffer, twice in kinase buffer (20 mM Tris
pH 7.5, 20 mM NaCl, 1 mM EDTA, 3% glycerol, 10 mM
MgCl2, 10 mM CaCl2, 1 mM
PMSF) and then incubated for 40 min in 25 µl of kinase buffer
supplemented with 1.5 µg of myelin basic protein (Sigma) and 10 µCi
of [-32P]ATP (Amersham Pharmacia Biotech) at
37°C. Samples were boiled for 4 min in SDS-PAGE loading buffer
containing 2 mM DTT and resolved on a 12% SDS-PAGE gel, after which
the gel was dried and subjected to autoradiography. To measure total
IRAK protein, lysates (20 µl) were resolved by 10% SDS-PAGE under
reducing conditions, transferred to a nitrocellulose membrane, and
Western blotted with the anti IRAK pAb (1:1000 in PBS, 3% BSA, and
0.1% Tween 20) used for the immunoprecipitations. Specific IRAK bands
were detected using a HRP-labeled goat anti-rabbit pAb and the ECL
chemiluminescence system (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain DC for TLR analysis, monocytes were cultured with GM-CSF
and IL-4, and by day 6 a homogeneous population of iDC was
recovered based upon expression of defining surface markers including
high amounts of CD1a, low levels of CD86, and practically no CD14 or
CD64 (Fig. 1A). In parallel,
cells were examined for TLR message by Northern analysis. Fig. 1B shows that fresh monocytes express high levels of TLRs 1,
2, 4, and 5, all of which decreased as monocytes differentiated into
iDC. Changes were apparent as early as the second day of culture, and
were complete at day 4, by which time low levels of TLRs 1, 2, and 4
were observed, whereas TLR5 was barely detectable. Because TLR6, an
isoform of TLR1, would also be detected by our TLR1 probe, we hereafter
refer to bands recognized by this probe as TLR1/6. The only TLR to
increase during iDC formation was TLR3, which was undetectable in
monocytes but clearly discernable as a band in iDC. -actin, which
was used as a control for RNA integrity, increased during iDC
formation, indicating that TLR down-regulation in iDC was not an
artifact of message degradation.
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Next, TLR message expression was examined in mDC. DC maturation is
induced by PAMPs such as LPS by inflammatory cytokines, such as
TNF-, and by CD40 ligand. FACS and functional analyses (Fig. 2, A and B and data
not shown) confirmed that LPS and TNF- did induce DC maturation as
indicated by marked increases in CD80, CD83, CD86, and HLA-DR and by
enhanced Ag-presenting capacity. Northern analyses revealed that all
TLRs with the exception of TLR1/6 were strongly repressed in mDC (Fig. 2C, iDC + LPS 24 h or iDC + TNF- 24 h), whereas
TLR1/6 was expressed at levels comparable to those seen in iDC.
However, to reach the low levels of TLR expression seen in mDC, the DC
passed through an intermediate with increased levels of TLRs 1/6, 2,
and 4 (Fig. 2C, 5-h treatment with LPS or TNF-). We then
focused on the early time points of expression of TLRs 2 and 4,
putative signaling receptors for LPS, following stimulation with LPS.
The pattern shown in Fig. 2D reveals a coordinated
up-regulation of the two TLRs that peaked at 3 h, and declined
rapidly thereafter. Thus, TLR expression defines an intermediate in the
DC maturation pathway in which some TLRs are expressed at relatively
high levels, before their down-regulation in mDC.
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shows that LPS induced changes in TLR
expression patterns in monocytes that were quite distinct from those
seen in iDC (compare with Fig. 2
). Monocytes, in contrast to iDC, did
not respond to prolonged (24-h) LPS treatment by down-regulating TLR
message. This effect was particularly apparent in TLRs 1/6, 2, 4, and
5, which were highly expressed in monocytes stimulated for 24 h
but not in mDC. The effects of short-term (2- or 4-h) LPS stimulation
fell into two patterns. In the first pattern, seen in TLRs 1/6 and 5,
LPS induced a dramatic decrease in message as early as 2 h,
followed by a return to either pretreatment or higher levels by 24
h. In the second pattern, seen in TLRs 2 and 4, rapid increases in
message were followed by a return to near prestimulation levels.
Interestingly, TLR1/6 changed from the first pattern in monocytes, to
the second pattern in iDC. These results show that levels of TLR
expression are regulated in a cell type-specific manner and
independently, except for TLRs 2 and 4, which were coordinately
expressed in all cells tested.
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To correlate message levels with surface protein expression, we
stained cells with mAbs against TLRs 1 and 4. FACS analysis (Fig. 4, A and B) showed
that TLR1 is easily detected on monocyte cell surfaces, but that the
level of surface expression drops by 10-fold in iDC and another
2-fold in mDC, where it is just barely detectable. TLR4 was present on
monocytes at easily detectable levels but was too low to detect by FACS
in iDC and mDC. The numbers of molecules of TLRs expressed on monocytes
were estimated by comparing the fluorescent signal from an
anti-CD44 mAb with those from the mAbs against TLRs 1 and 4. In
these experiments, all mAbs were mouse IgG1, and the same secondary
reagent was used in staining. Using radioiodinated anti-CD44, we
estimated that 1.5 x 105 mAb molecules
bound per cell at saturation (corresponding to twice as many CD44
molecules, if the mAb bound divalently; Ref. 37). It is
apparent in Table II that surface
expression of both TLRs 1 and 4 is extremely low compared with CD44 (an
adhesion molecule), corresponding to a few thousand molecules per cell
on monocytes, and a few hundred molecules or less on iDC. Moreover,
surface expression of both TLRs 1 and 4 showed high donor variability.
For example, monocytes from one donor expressed 400 TLR4
molecules/cell, whereas another expressed 3200, with the remaining
donors distributed more or less evenly in between. Monocytes from two
donors failed to stain with the anti-TLR1 mAb, and the remaining 13
ranged from 400 to 5400 molecules/cell (Table II).
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We next asked whether TLRs might play a role in LPS-induced signal
transduction in iDC. TLR4, which is essential for LPS signaling in
mice, requires a second protein, MD2, for optimal LPS responses, so we
first probed monocytes, iDC, and mDC for its expression (Fig. 5). Interestingly, MD2 is expressed at
significantly higher levels in iDC than in monocytes, leading to an
inversion of the MD2/TLR4 ratio in these two cell types. For
comparison, CD14 message is totally lacking in iDC (Fig. 5) as expected
from its lack of surface expression (Fig. 1A). CD14 is known
to be required for LPS responsiveness (39), and in our
studies is supplied in soluble form from the serum. Thus, although
surface expression of TLR4 is exceptionally low, all components known
to be required for a functional TLR4 are present in iDC. Because iDC
lose expression of TLRs 2, 3, 4, and MD2 during maturation, we would
predict that if any of these is essential for LPS signaling, then mDC
should not respond to LPS. We tested this by measuring TNF-
secretion. As shown in Fig. 6A, LPS triggered a robust
TNF- response in iDC but failed to induce TNF- secretion in mDC,
even though mDC were capable of responding to PMA plus ionomycin. A
second prediction of TLR signaling is that LPS should induce IRAK
activation in iDC but not mDC. IRAK is the first kinase to be activated
by TLRs, IL-1R, and IL-18R, but is not known to be activated by any
other receptor. Fig. 6B shows that LPS does in fact induce
IRAK activation in iDC, but not in mDC as measured in a kinase assay,
although both cell types express similar amounts of IRAK as seen in the
anti-IRAK Western blot. Activation was not due to a secondary
effect of IL-1 induction, because a neutralizing anti-IL-1 mAb
failed to block LPS-induced activation of IRAK, and similar results
were obtained with an anti-IL-18 mAb (data not shown). Thus, our
data strongly suggest that LPS activates iDC through one or more TLRs.
However, we have not yet been able to determine precisely which TLRs
are involved because none of the anti-TLR Abs available to us
blocks LPS signaling in iDC (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and
IRAK activation. After submission of this manuscript, another paper
examining TLR message expression was published by Muzio et al.
(38). Their results are in general agreement with ours,
except for TLR5, which they find expressed in DC at levels even higher
than in monocytes. The reason for this discrepancy is not known, but
may be due to differences in the methods of DC preparation. At the level of surface expression, TLRs 1 and 4, the two TLRs for which we had mAbs, were expressed in low numbers on monocytes and, as expected from the Northern analyses, even lower numbers on iDC. TLR1 protein is barely detectable by FACS on iDC and mDC, whereas TLR4 cannot be detected, but may be present in very small amounts on iDC because these cells make TLR4 message. Du et al. (40) previously reported that mouse macrophage lines express amounts of TLR4 protein that are so low that they are limiting for LPS responsiveness, meaning that increases in TLR4 expression resulted in increases in LPS responsiveness. Therefore, the low expression of TLRs 1 and 4 that we observed in normal cells is likely to be an important aspect of TLR function because under limiting conditions, cellular responses to PAMPs could be stringently regulated by controlling the amounts of TLR protein produced. Moreover, the exceptionally high variability in TLR surface expression that we observed among normal donors might indicate a high variability in the way different individuals respond to PAMPs. However, because monocytes exhibit relatively homogeneous distributions of TLRs 1 and 4, it is unlikely that subpopulations of differentially reactive cells exist within an individual. Another factor that could control TLR4 function is MD2, a molecule that associates with the extracellular portion of TR4 and is required for maximal TLR4 function (19). The fact that the MD2/TLR4 ratios are inverted in monocytes and iDC suggests that MD2 might be limiting for the LPS response in monocytes, whereas TLR4 would be limiting in iDC. However, there might be other ramifications of the relatively high MD2 expression in iDC, for example, MD2 might be secreted from iDC, it might bind to other iDC-specific TLRs (e.g., TLR3), or it might compensate in some way for the lack of CD14 surface expression in iDC.
We have observed that LPS stimulation regulates the expression of all five TLRs in monocytes and iDC, but the TLRs are regulated differently in the two cell types. Two TLRs, 2 and 4, are required for responses to a number of bacterial products, and it is of interest that these two TLRs are coordinately regulated in both monocytes and iDC, perhaps reflecting their similar functions. Although it has not been proven conclusively, a consensus is emerging that TLR4 is the sole signal-transducing receptor for LPS in the human as it is in mice, and that TLR2 is a receptor for several other bacterial PAMPs (41). If this is true, then one important question arising from our results is whether TLR4 is expressed on iDC in sufficient amounts to account for the LPS response. We estimate that iDC express at most 150 TLR4 molecules per cell, and expression could be considerably less, even zero. In the case of the IL-1R, which shares homology with the TLRs in the cytoplasmic signaling domains, 10 or fewer ligated receptors can induce a response (42), thus providing a precedent for signaling by extremely low numbers of receptors in a closely related system. Alternatively, iDC may not express TLR4 molecules on their surfaces at all, and either LPS or a bacterial contaminant may have activated TLR2 or a different TLR in our experiments. A third possibility is that TLRs are expressed primarily in intracellular compartments, and function by interacting with internalized PAMPs. We are currently investigating these possibilities. Regardless of TLR specificity, the observations that LPS activates IRAK, and that the ability of LPS to trigger iDC function parallels TLR expression, provide strong evidence that PAMPs signal through TLR-dependent pathways in iDC. By inducing iDC maturation, these TLR-dependent signals could play a pivotal role in the development of adaptive responses to pathogens.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David Segal, Experimental Immunology Branch, National Cancer Institute, Building 10, Room 4B36, National Institutes of Health, Bethesda, MD 20892-1360.
3 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; mDC, mature DC; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; IRAK, IL-1 receptor-associated kinase.
Received for publication May 10, 2000. Accepted for publication October 6, 2000.
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A. Karlsson, E. Nygren, J. Karlsson, I. Nordstrom, C. Dahlgren, and K. Eriksson Ability of Monocyte-Derived Dendritic Cells To Secrete Oxygen Radicals in Response to Formyl Peptide Receptor Family Agonists Compared to That of Myeloid and Plasmacytoid Dendritic Cells Clin. Vaccine Immunol., March 1, 2007; 14(3): 328 - 330. [Abstract] [Full Text] [PDF]
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S. Fernandez, D. R. Palmer, M. Simmons, P. Sun, J. Bisbing, S. McClain, S. Mani, T. Burgess, V. Gunther, and W. Sun Potential Role for Toll-Like Receptor 4 in Mediating Escherichia coli Maltose-Binding Protein Activation of Dendritic Cells Infect. Immun., March 1, 2007; 75(3): 1359 - 1363. [Abstract] [Full Text] [PDF]
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 H. Bartz, N. M. Avalos, A. Baetz, K. Heeg, and A. H. Dalpke Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation Blood, December 15, 2006; 108(13): 4102 - 4108. [Abstract] [Full Text] [PDF]
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R. Yadav, D. J. Zammit, L. Lefrancois, and A. T. Vella Effects of LPS-mediated bystander activation in the innate immune system J. Leukoc. Biol., December 1, 2006; 80(6): 1251 - 1261. [Abstract] [Full Text] [PDF]
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) were irradiated and added to
1 x 105 responder allogeneic T cells.
[3H]thymidine incorporation was measured after 3 days.
Background T cell proliferation was <500 cpm. C,
Northern analysis of TLR expression in monocytes, iDC, and mDC. As
indicated, mDC were generated by incubating iDC for 24 h with
either LPS or TNF-
