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Institute of Cancer Research and Molecular Biology, The Norwegian University of Science and Technology, N-7489 Trondheim, Norway
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and TNF. On purified
granulocytes, LPS, GM-CSF, and TNF down-regulated, and IL-10 modestly
increased TLR2 expression after 2 h. These data suggest that TLR2
protein expression in innate immune cells is differentially regulated
by inflammatory mediators.
Key Words: monocytes • granulocytes • innate immunity cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blotting has shown expression of TLR2 in most lymphoid tissues, with the highest expression seen in peripheral blood leukocytes, but also in brain, lung, muscle, and heart tissue [2 , 16 , 17 ]. TLR2 expression has been demonstrated in monocytes and macrophages [13 , 17 ], polymorphonuclear cells [18 ], dendritic cells [18 ], endothelial cells [19 ], epithelial cell lines [20 ], and murine T-cells [16 ]. The presence of TLR2 in primary human cells has been studied mostly on mRNA level with reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blotting, however a definitive quantitative analysis of TLR2 protein expression has not been shown. We have used two anti-TLR2 mAbs developed in our laboratory to examine protein expression and surface regulation of TLR2 in human cell lines and primary cells, as well as in human lymphoid tissues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(GlaxoWellcome, London, UK); rIL-4
(Peprotech EC, London, UK); rIL-6 (Genzyme, Cambridge, MA); recombinant
tumor necrosis factor (rTNF; Genentech, South San Francisco, CA);
rIL-10, recombinant interferon- (rIFN-), and recombinant
transforming growth factor-ß (rTGF-ß; R&D Systems, Minneapolis,
MN); recombinant granulocyte-macrophage colony-stimulating factor
(rGM-CSF; Sandoz, Basel, Switzerland); and recombinant TNF-related
activation-induced cytokine (rTRANCE) and rM-CSF (RDI Inc.,
Flanders, NJ). A new TLR2-specific mAb, TL2.3, was developed in our
laboratory as previously described for the mAb TL2.1
[13
]. TL2.3 showed identical staining specificity as
TL2.1 on peripheral blood mononuclear cells (PBMC) and transfected cell
lines (unpublished results). The TL2.1 mAb and a control mouse
immunoglobulin G (IgG; Caltag Laboratories, Burlingame, CA) were
labeled with Alexa FluorTM 488 (A488) fluorescent dye, as described by
the manufacturer (Molecular Probes, Eugene, OR). Other mAbs toward the
following antigens were used: CD3, CD14 (LeuM3), CD19
(Becton-Dickinson, Mountain View, CA), CD14 (18D11; Diatec, Oslo,
Norway), CD15 (Sigma Chemical Co.), and CD56 (Exalpha Biologicals,
Boston, MA).
Cells
PBMC were isolated from human A+ buffycoats (The Bloodbank, RiT,
Trondheim, Norway) with Lymphoprep, as described by the manufacturer
(Nycomed Amersham, Oslo, Norway). Monocytes were isolated by adherence
to plastic (60 min, 37°C) in RPMI supplemented with 5% pooled human
A+ serum (The Bloodbank). Granulocytes were isolated from heparinized
whole blood with Polymorphprep as described by the manufacturer
(Nycomed Amersham). In some experiments, purified monocytes and
granulocytes were incubated with LPS (100 ng/ml), IL-1 (100 ng/ml),
IL-4 (100 U/ml), IL-6 (100 ng/ml), IL-10 (100 U/ml), TNF (10 ng/ml),
IFN- (100 U/ml), GM-CSF (100 U/ml), or TGF-ß (100 ng/ml) for 2–20
h before analysis of TLR2 and CD14 expression. In parallel, monocytes
were preincubated with the cytokines for 20 h prior to stimulation
with synthetic bacterial lipohexapeptide 47L (BLHP) from
Treponema pallidum [11
]. Incubation proceeded
for 8 h at 37°C before supernatants were assayed for
TNF-activity in the WEHI 164 clone 13 bioassay [21
].
CD56+ natural killer (NK) cells were isolated immunomagnetically using anti-CD56 (Leu-19; Becton-Dickinson)-coated PBMC and anti-mouse IgG2a-coated M450 Dynabeads (Dynal, Oslo, Norway) at bead-to-PBMC ratio 1:1, as previously described [22 ]. T- and B-cells were isolated with anti-CD3-coated or anti-CD19-coated Dynabeads (Dynal), respectively, at bead-to-PBMC ratio 1:1, as described by the manufacturer. The purity of T-, B-, and NK cells was always >95%, as determined by flow cytometry. Macrophages were 4–7-day-old monocytes [23 ], and osteoclasts were multinucleated cells obtained by culturing monocytes for 7 days in the presence of TRANCE (50 ng/ml) and M-CSF (50 ng/ml) [24 ]. All primary cells were grown in RPMI/5% A+ serum.
The following cell lines were generous gifts: U373, from Dr. Guillemont
(Santi, France); human endothelial (HMEC)-1, from Dr. M. Arditi
(University of California, Los Angeles, CA); Mono Mac 6, from Dr.
H. W. Ziegler-Heitbrock (Institute of Immunology, Munich,
Germany); KYM-1, from Dr. A. Meager (The National Institute for
Biological Standards and Control, South Mimms, UK); and FS4, from Dr.
J. Vil
ek (New York School of Medicine, New York, NY). All other
cell lines were from the American Type Culture Collection (ATCC,
Rockville, MD).
Tissue staining
Frozen sections of human tonsils, lymph nodes, and appendices
were fixed in acetone and incubated with 3%
H2O2 for blockade of endogenous peroxidase and
with anti-TLR2 mAb (TL2.3) for 30 min at room temperature. The optimal
working dilution of the antibody was estimated by titration. A standard
avidin-biotin peroxidase technique was used. AEC
(amino-ethyl-carbazole) was used as chromogene, and the sections were
counterstained with haematoxylin. The immunostainings were done using
an immunohistostainer (DAKO Techmate 500, Dakopatts A/S, Glostrup,
Denmark). In each staining run, negative controls were included where
the primary antibody was omitted.
Cell staining
Whole blood (100 µl) was incubated with mAbs (10 µg/ml, 30
min, 4°C), red cells were lysed with formic acid, pH was adjusted
with ammonium chloride, and cells were fixed in 2% paraformaldehyde on
a MultiQprep (Beckman Coulter, Fullerton, CA) according to
instructions. Extracellular labeling was done in phosphate-buffered
saline (PBS)/0.1% bovine serum albumin (BSA; cell lines) or PBS/2%
pooled human serum (primary cells) with the indicated Abs (10 µg/ml,
30 min, 4°C). Dead cells staining positive for propidium iodide (2.5
µg/ml) were gated out of analysis. For intracellular staining, cells
were fixed (PBS/2% formalin, 10 min, 4°C), permeabilized, and
blocked for unspecific binding (PBS/20% pooled human serum/0.1%
saponin, 20 min, 20°C), stained with primary mAbs (10 µg/ml, 30
min, 20°C), washed twice, incubated with secondary goat anti-mouse
Ig-fluorescein isothiocyanate (GAM-FITC; Becton-Dickinson; 10 µg/ml,
30 min, 20°C), washed three times, and analyzed. Cells prepared for
confocal microscopy (LSM 510; Zeiss, Jena, Germany) were fixed (PBS/2%
formalin, 15 min, 4°C), permeabilized (acetone, 10 min, -20°C),
stained with anti-TLR2-A488 (TL2.1) and anti-CD14-phycoerythrin (PE;
18D11) mAbs (5–10 µg/ml, 60 min, 20°C) in PBS/10% fetal calf
serum (FCS)/0.1% BSA, and washed three times.
Metabolic labeling
Monocytes and T-, B-, and NK cells were incubated with 100
µCi/ml TRAN35S-LABEL (ICN, Costa Mesa, CA) in methionine-
and cysteine-free RPMI (ICN) containing 10% dialyzed FCS for 16 h
at 37°C in the presence or absence of 5 ng/ml PMA + 500 ng/ml
ionomycin. The cells were lysed, immunoprecipitated with anti-TLR2 mAb
(TL2.1)-conjugated Sepharose, and separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) exactly as
described [13
].
RT-PCR
RNA was isolated from 2 x 106 monocytes and
T-, B-, and NK cells with the High Pure mRNA isolation kit (Boehringer
Mannheim, Mannheim, Germany), as described by the manufacturer. RT-PCR
was performed as described [25
]. TLR2 mRNA fragments
were PCR-amplified with the primer pair 5'-AGGCAAAATCATTTGGCA-3' and
5'-CTTTGGCCAGTGCTTGCT-3', selected on the basis of the published human
TLR2 sequence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whole blood double-stained with TL2.1-A488 and PE-conjugated, cell-specific mAbs (CD3, CD14, CD15, and CD56) showed that CD14+ monocytes expressed the highest levels of TLR2, followed by CD15+ granulocytes, and that CD3+ T-cells and CD56+ NK cells did not express detectable levels of TLR2 (Fig. 1 ). Moreover, highly purified CD3+ T-cells, CD19+ B-cells, and CD56+ NK cells did not express TLR2 on the surface or intracellularly, even when stimulated with PMA and ionomycin or cytokines (unpublished results). The results were confirmed by immunoprecipitation of metabolic [S35]-labeled cell lysates with the TL2.1 mAb. Again, expression of TLR2 was undetectable in unstimulated and PMA/ionomycin-stimulated (16 h) lymphoid cells, and monocytes expressed high levels of TLR2 (Fig. 2 ). However, by using RT-PCR, low levels of TLR2 mRNA were found in T-, B-, and NK cells (unpublished results); thus, we cannot rule out the possibility of a very low level of TLR2 expression in peripheral blood lymphoid cells.
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, and TGF-ß (Fig. 5A and
unpublished results). The basal level of TLR2 on unstimulated
granulocytes had decreased after 20 h. TNF further down-regulated
TLR2, whereas the other cytokines were without effect. Granulocyte CD14
was regulated similarly to TLR2 after 2 h (Fig. 5B)
, but contrary
to TLR2, CD14 was up-regulated by LPS, GM-CSF, IL-1, and IL-4 after
20 h. No apparent effects were seen with IL-6, IFN-, and
TGF-ß at any time points (unpublished results). Thus, whereas the
level of granulocyte TLR2 was decreased rapidly by LPS and GM-CSF and
remained low, LPS and GM-CSF induced only transient down-regulation of
CD14 expression (at 2 h) followed by up-regulation after 20 h.
LPS, GM-CSF, IL-1, and IL-10 all markedly increased TLR2 expression on
monocytes, and IL-4 down-regulated TLR2 potently after a 20-h
incubation (Fig. 6A)
. Modest down-regulation was seen with IFN- and
TNF, whereas IL-6 and TGF-ß did not modulate the TLR2 levels (not
shown). After 2 h, only minor changes in TLR2 expression were
seen. The regulation of TLR2 expression was paralleled by CD14 (Fig. 6B)
, suggesting similar mechanisms of regulation of these proteins in
monocytes.
We further examined if the cytokine-induced up- or down-regulation of
TLR2 coincided with an increased or decreased TLR2-mediated activation
of monocytes. Monocytes were preincubated with various cytokines for
20 h prior to stimulation with the TLR2 ligand, synthetic BLHP
from T. pallidum [11
]. IFN- and GM-CSF
increased TNF production induced by BLHP (unpublished results) but
affected the TLR2 level in opposite directions (Fig. 6A)
. Moreover,
TGF-ß reduced, and IL-4 and IL-10 blocked BLHP-induced TNF production
(not shown), and the TLR2 level was unaffected, down-regulated, and
up-regulated, respectively (Fig. 6A)
. The effects of IL-1 and TNF were
difficult to interpret, because these cytokines induced high-background
TNF production. Thus, no apparent correlation was found between the
surface TLR2 levels and the TNF production in response to BLHP in the
monocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Little information is available on TLR2 expression and regulation on the protein level. In this study, we show that TLR2 expression in blood cells is confined to CD14+ monocytes and CD15+ granulocytes with the lymphocytes being TLR2-negative, which is in agreement with TLR2 mRNA data presented by Muzio et al. [18 ]. Although the TLR2 protein was undetectable in resting and PMA/ionomycin-activated lymphocytes, RT-PCR showed the presence of TLR2 mRNA in purified T-, B-, and NK cells (unpublished results). Thus, the results from Matsuguchi et al. [16 ], showing that murine T-cells contain TLR2 mRNA, are not in conflict with the present study, and we cannot rule out the possibility that human lymphocytes may express TLR2 protein at a level below the detection limit of our TLR2 mAbs. The finding that TLR2 was detected in activated but not in resting germinal-center B-cells in lymphoid tissues may indicate that TLR2 is expressed only at certain differential stages or in subpopulations of lymphocytes.
In vitro-differentiated monocytes showed membrane staining of TLR2 as well as staining associated with intracellular vesicles and the nucleus. The intracellular vesicles were often found to contain CD14 and TLR2. These vesicles are likely to represent post-Golgi carriers, because they did not stain positive for wheat germ agglutinin (WGA), which binds to the Golgi apparatus (not shown). The nuclear staining of TLR2 in monocytes, monocyte-derived macrophages, and osteoclasts suggests direct import of TLR2 to the nucleus [30 ] or that TLR2 is co-transported by an associated protein into the nucleus, as seen for the IL-1 receptor [31 ]. The nuclear localization of TLR2 is being investigated currently.
Muzio et al. [18
] showed recently that LPS
increased TLR4 mRNA but not TLR2 mRNA in human monocytes. However,
Medvedev and colleagues [32
] found rapid (<1 h)
induction of TLR2 mRNA in mouse macrophages stimulated with LPS. In
addition, LPS induces TLR2 mRNA transcription in HMEC-1 in a nuclear
factor-
B (NF-B)-dependent manner [33
]. Our own
unpublished results also show that 24 h of LPS stimulation
increases the TLR2 mRNA levels in monocytes (unpublished results). On
the protein level, we found that surface TLR2 on monocytes was not
affected significantly by LPS after 2 h, whereas the TLR2 levels
increased markedly after longer exposure (20 h). This agrees with a
recent study showing that in the adipocyte, newly synthesized TLR2 is
processed extensively with an intracellular half-time of 3–3.5 h
before reaching the surface [34
]. Thus, LPS-induced,
surface TLR2 seems to be regulated at the transcriptional level in
monocytes.
Recently, it was shown that IL-4 reduced the TLR2 mRNA level in human monocytes after 36 h, and IL-10 was without effect [35 ]. We observed a corresponding down-regulation of monocyte-surface TLR2 after 20 h with IL-4 but also that IL-10 increased the expression of TLR2. Hence, it seems that LPS and IL-4, but not IL-10, regulate TLR2 at the transcriptional level.
On granulocytes, surface TLR2 was down-regulated by LPS after 2 h and remained low. This seems contradictory to the findings by Muzio et al. [18 ] that LPS increased TLR2 mRNA in polymorphonuclear cells after 3 h. It may be that the mRNA level does not necessarily correlate with surface expression of TLR2, as shown for TLR4 in murine peritoneal macrophages; whereas TLR4 mRNA only transiently decreased in response to LPS, the membrane protein remained down-regulated for 24 h [36 ]. The relatively short period for granulocyte TLR2 down-regulation also suggests that mechanisms other than transcriptional regulation are important determinants of the protein level. The mode of action may include externalization of pre-made TLR2, protein shedding, or internalization, as shown for CD14 [37 , 38 ]. We have preliminary data showing the presence of soluble TLR2 in serum and in monocyte supernatants. An enzyme-linked immunosorbent assay (ELISA) specific for TLR2 is under development to investigate this further. The level of TLR2 expression will also depend on the stability of the protein, and, therefore, we stress the importance of confirming mRNA data by measuring the actual protein expression.
In the present study, CD14 and TLR2 had similar regulation patterns in the monocyte in response to LPS or cytokines. CD14 and TLR2 were also similarly affected in granulocytes after 2 h, but after 20 h, LPS, IL-1, IL-4, and GM-CSF all up-regulated CD14, whereas no difference from the basal level was observed for TLR2. Thus, after 20 h, the CD14 regulatory effects of LPS, IL-1, and GM-CSF were similar in monocytes and granulocytes, whereas IL-4 increased granulocyte CD14 and decreased monocyte CD14. Surprisingly, IL-10 increased TLR2 and CD14 expression consequently in monocytes and granulocytes.
IL-4 and IL-10 are regarded as anti-inflammatory cytokines, because
they inhibit the release of pro-inflammatory cytokines, although
through different mechanisms [39
40
41
42
]. We found that
IL-4 and IL-10 blocked monocyte TNF production induced by the TLR2
ligand BLHP from T. pallidum [11
]
(unpublished results). The down-regulation of monocyte TLR2 and CD14
expression by IL-4 could explain the anti-inflammatory effects
observed. However, additional intracellular mechanisms like enhanced
mRNA degradation may also be involved [39
]. From our
data, it is apparent that the anti-inflammatory effects of IL-10 cannot
be explained by regulation of membrane TLR2 and CD14 expression. The
inhibitory effect may rather occur at the level of gene activation,
because IL-10 has been shown to inhibit activation of NF-B
[39
].
The co-regulation of TLR2 and CD14 in monocytes (20 h) and granulocytes (2 h) suggests that similar mechanisms are responsible for transcriptional activation. Consensus motifs for the transcription factors Sp1, PU.1, Myb, AP-1, AP-2, and CDP are located 5' upstream to human CD14 [43 ]. The upstream sequence has not been published for human TLR2, but in murine T-cells, TLR2 regulation is dependent on extracellular-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase and is partly dependent on Ras [16 ]. More sequence data are needed to examine this issue further.
Taken together, we have shown that monocytes and granulocytes regulate TLR2 expression in response to LPS and cytokines. LPS and GM-CSF increased TLR2 on monocytes after 20 h and rapidly reduced surface levels on granulocytes within 2 h. This difference in regulation may reflect that granulocytes are the first line of defense toward the invading microbe and that monocytes are recruited and activated later than the granulocytes. How TLR2 expression is regulated on the innate immune cells during disease is currently studied in our laboratory.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received August 7, 2000; revised October 17, 2000; accepted October 20, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms J. Biol. Chem. 270,9558-9563Related Article
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