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Section of Functional Genomics, Division of Genomic 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and mitogen-activated protein kinases in a manner
similar to that of IL-1 (6, 7, 8). The related receptor
TLR2, originally reported as a receptor for LPS (9), is
probably principally involved in response to microbial lipoproteins
that often contaminate commercial LPS preparations (10),
although CD14 and MD-2 can facilitate repurified LPS signaling
via this receptor (11). Signaling via TLR2 is more
complex, and is at least partly dependent upon heterodimerization of
this receptor with either TLR1 or TLR6 (12, 13, 14). Dependent upon dose and route of exposure, LPS can cause or be associated with septic shock, the exacerbation of allergic inflammation (e.g., in asthma) (15, 16), and immune deviation from Th2 phenotypes to Th1 phenotypes (17). At a cellular level, the multiple functions of LPS and bacterial lipoproteins include the priming of responses to inflammatory mediators (18, 19, 20), cell activation (1), proliferation (21), and both the inhibition (22) and induction of apoptosis (23). However, there is still uncertainty about which peripheral blood leukocyte types respond to LPS.
The responses of monocytes to LPS include induction of cytokine synthesis (24), with concomitant effects on the survival, proliferation, and immune deviation of other cell types. Neutrophils are the other primary leukocyte type involved in protection of the host from bacterial invasion and have long been held to be sensitive to LPS, resulting in modulation of adhesion molecule expression, cytokine generation, and cell life span (1, 18, 22, 25). However, gradient-based cell preparation techniques almost invariably leave a low level of monocyte contamination of neutrophilpreparations. Eosinophils have been recently reported to be LPS responsive and to express CD14 protein and TLR2 and TLR4 mRNAs (26), but a contradictory report found them to be CD14 negative and their apparent LPS responsiveness to be dependent upon the presence of monocytes (27). Basophils have been shown to be LPS responsive, but again in cell suspensions not fully depleted of CD14+ monocytes (28, 29), and their patterns of TLR expression are wholly unknown. Therefore, we set out to investigate whether patterns of TLR mRNA and protein expression on these cell types correlated with patterns with LPS responsiveness in standard gradient-purified preparations and after further purification by negative selection to remove contaminating monocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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General laboratory reagents were from Sigma (Poole, U.K.). LPS from Escherichia coli serotype 0111:B4 was from Sigma. Repurified LPS (10) was a generous gift from Dr. S. Vogel (Uniformed Services University of Health Sciences, Bethesda, MD). Synthetic bacterial lipopeptide Pam3CysSerLys4 was from EMC Microcollections (Tübingen, Germany). PE-conjugated anti-TLR4 mAb (clone HTA125, isotype IgG2a), PE-conjugated anti-TLR2 mAb (clone TL2.1, isotype IgG2a), PE-conjugated anti-CD14 mAb (clone 61D3), PE-conjugated anti-CD11b, FITC-conjugated anti-L-selectin, and isotype controls were from eBioscience (San Diego, CA). Cytokines and chemokines were from PeproTech (London, U.K.). FCS and PBS were from Life Technologies (Paisley, U.K.). All experiments were performed using a lot of FCS with known extremely low endotoxin levels (0.371 ng/ml, contributing <0.01 ng/ml endotoxin when used at 2% in our assay buffers). HotStar Taq and mini-RNeasy purification kits were purchased from Qiagen (Crawley, U.K.), dNTPs were purchased from Hybaid (Ashford, U.K.), Moloney murine leukemia virus H- reverse transcriptase (RT) and RNAsin from Promega (Southampton, U.K.). PCR primer pairs and real-time probes were designed using MacVector software (Accelrys, Cambridge, U.K.). PCR primer pairs were based on areas of TLRs showing least homology to each other and were purchased from Sigma Genosys (Cambridge, U.K.) and MWG Biotech (Ebersberg, Germany); real-time PCR primers and dual-labeled oligonucleotide probes were from MWG Biotech.
Cell preparation
Peripheral venous blood was taken with informed consent from normal volunteers in accordance with a protocol approved by the South Sheffield Research Ethics Committee. Blood was anticoagulated with trisodium citrate, plasma, and platelets removed by centrifugation, and following dextran sedimentation PBMC were separated from granulocytes by density over a plasma/Percoll (Amersham Pharmacia, St. Albans, U.K.) gradient as described (30), using a method developed to produce nonactivated leukocytes suitable for study of LPS responses (30, 31). In some experiments, leukocyte populations were further purified by negative magnetic selection (32). Ab mixtures were purchased from StemCell Technologies (Vancouver, Canada). Eosinophils were purified from granulocytes using a mixture containing Abs to CD2, CD14, CD16, CD19, CD56, and glycophorin A. Basophils were purified from PBMC using a mixture containing Abs to CD2, CD3, CD14, CD15, CD16, CD19, CD24, CD34, CD36, CD45RA, CD56, and glycophorin A. Monocyte depletion of granulocyte or PBMC preparations was achieved using an anti-CD14 custom mixture, or a custom mixture containing Abs to CD36, CD2, CD3, CD19, CD56, and glycophorin A. Briefly, cells were incubated in buffer (PBS without Ca2+/Mg2+, 2% FCS, and 10 mM HEPES (pH 7.3–7.4)) with the relevant Abs and magnetic colloid according to manufacturer’s instructions (at room temperature for all purifications except eosinophils, which were incubated with Ab mixtures on ice), applied to a sterile column containing metal mesh and separated in a magnetic field, and eluted from the column in buffer containing 1 mM EDTA (32). Purified cells were washed into the appropriate assay buffer and counted using a hemocytometer.
Modulation of cell surface marker expression
Leukocytes were resuspended at 5 x 106 cells/ml in assay buffer (Dulbecco’s modified PBS containing Ca2+/Mg2+, 2% FCS, 10 mM HEPES, and 0.18% glucose (pH 7.3–7.4)) and stimulated for 1 h at 37°C in 50-µl aliquots with buffer or agonists. Control samples treated with buffer alone provided baseline levels. Following stimulation, all samples were washed with ice-cold FACS buffer (PBS without Ca2+/Mg2+, 10 mM HEPES, and 0.25% BSA (pH 7.3–7.4)), pelleted by centrifugation (1000 x g for 2 min at 4°C), and stained with the relevant Abs (see Flow cytometry). L-Selectin expression and CD11b expression levels were all quantified as the percentage of basal values using the geometric mean of their fluorescence, except for neutrophil L-selectin expression, where cells formed a bimodal distribution of high and low L-selectin expression. Thus, for neutrophils, L-selectin levels were quantified in terms of percentage of cells showing high expression before and after stimulation.
Flow cytometry
Leukocytes were resuspended at 5 x 106 cells/ml in FACS buffer (see Modulation of cell surface marker expression) and 50-µl aliquots stained with appropriate Abs and matched isotype controls by incubation on ice for 30 min, washed in ice-cold FACS buffer, pelleted by centrifugation (1000 x g for 2 min at 4°C), and resuspended in PBS. Ab dilutions were as follows: PE-anti-CD11b and FITC-anti-L-selectin, 1/25; PE-anti-TLR2, PE-anti-TLR4, and PE-anti-CD14, 1/10; FITC-anti-HLA-DR (Sigma), 1/50; PE-anti-CD123, 1/50; and biotin-anti-CD123, 0.3 µg/ml. To minimize nonspecific binding of anti-TLR mAbs, incubation was conducted in the presence of 50 µg/ml mouse IgG (Sigma). To separate neutrophils and eosinophils in mixed granulocyte preparations, cells were double-stained with anti-VLA-4 FITC (Serotec, Oxford, U.K.). To separate basophils in mixed PBMC populations, cells were stained with anti-HLA-DR FITC and anti-CD123-biotin concurrently with the other primary Abs, washed once, and stained with allophycocyanin-streptavidin (0.15 µg/ml; eBioscience). Flow cytometry was performed using a dual-laser FACSCalibur (BD Biosciences, Mountain View, CA) using CellQuest software (BD Biosciences), with appropriate single-stained samples for setting of compensation. To investigate TLR expression in whole blood, 100-µl aliquots of freshly sampled blood (anticoagulated with trisodium citrate) were incubated with PE-conjugated anti-TLR mAbs and isotype controls in the presence of 50 µg/ml mouse IgG and anti-VLA4-FITC as above for 30 min on ice. Samples were washed by the addition of 1 ml of FACS buffer, pelleted by centrifugation (1000 x g for 2 min), and resuspended in FACS buffer, the RBC were lysed, and leukocytes were fixed using Optilyse B (Beckman Coulter, Fullerton, CA) according to the manufacturer’s instructions. Optilyse B separates eosinophils and neutrophils on forward light scatter (FSC)/side light scatter (SSC) plots (33). Eosinophils and neutrophils were defined according to FSC/SSC gating combined with VLA-4 staining, and monocytes were defined by FSC/SSC gating.
Neutrophil survival
Granulocyte preparations were depleted of monocytes by CD14-negative selection under aseptic conditions. Aliquots of cells (2.5 x 106 cells/ml, 100-µl aliquots) were cultured in Falcon Flexiwell plates (BD Biosciences) with buffer or stimuli (LPS, in the presence or absence of autologous PBMC at either 1.5 x 106 or 1.5 x 105 cells/ml) in RPMI 1640, 10% FCS, penicillin, and streptomycin at 37°C in 5% CO2, as described previously (34, 35). After each time point replicates were pooled, washed in ice-cold FACS buffer, divided, and stained with anti-TLR Abs as described above, and cell viability was determined by vital dye staining in accordance with established techniques (36, 37) using ToPro-3 (1/10,000 dilution; Molecular Probes, Eugene, OR), an alternative to propidium iodide whose fluorescence is detectable in the FL-4 channel (38) (pilot data (not shown) demonstrated that To-Pro-3+ cells were all annexin V positive, consistent with their identity as a late apoptotic population (36, 37)). Granulocyte apoptosis was quantified by morphology on cytospins as described (34, 35).
RT-PCR
RNA was purified from aliquots of cells (
5 x
106 cells) using RNeasy kits according to the
manufacturer’s instructions. Contaminating genomic DNA was removed
using DNAfree (Ambion, Huntingdon, U.K.), and cDNA
was prepared from 2 µg total RNA using an RNase-H-
Moloney murine leukemia virus enzyme. RT-PCR of cDNA and non-RT
controls was performed using HotStar Taq according to the
manufacturer’s instructions over 35 cycles on a Hybaid PCR Express
(Hybaid), with the following primer pairs at their appropriate optimal
melting temperature as determined by MacVector software: TLR2
forward primer (5'-GGGTCATCATCAGCCTCTCC-3') and reverse primer
(5'-AGGTCACTGTTGCTAATGTAGGTG-3'); TLR4 forward primer
(5'-CAGAGTTGCTTTCAATGGCATC-3') and reverse primer
(5'-AGACTGTAATCAAGAACCTGGAGG-3'); CD14 forward primer
(5'-GGTGCCGCTGTGTAGGAAAGA-3') and reverse primer
(5'-GGTCCTCGAGCGTCAGTTCCT-3'); and MD-2 forward primer
(5'-GCTCAGAAGCAGTATTGGGTCTG-3') and reverse primer
(5'-CGCTTTGGAAGATTCATGGTG-3').
PCR products were analyzed by 1–2% agarose gel electrophoresis.
Real-time PCR
To quantify IL-8 mRNA generation, cDNA samples and their non-RT
controls were analyzed by real-time quantitative PCR. Purified
leukocyte populations (5 x 106 cells/ml,
50-µl aliquots) were stimulated with LPS or control stimuli for
1 h at 37°C in parallel with the experiments above
investigating L-selectin shedding/CD11b up-regulation. A total of 1
µl of cDNA or non-RT control (in duplicate) was amplified in 25 µl
using HotStar Taq in the presence of 3 mM
Mg2+ in an ABI 7700 thermal cycler (PE Applied
Biosystems, Foster City, CA), and fluorescence was monitored at each
cycle. Cycle parameters were 95°C for 15 min to activate
Taq followed by 40 cycles of 94°C for 15 s, 58°C
for 15 s, and 72°C for 30 s. Primers (final concentration,
1 µM), probes (final concentration, 200 nM), BSA (final
concentration, 250 µg/ml), and control DNA stocks were stored in
single-use aliquots. In each plate, target levels were quantified
against a standard curve constructed from serial dilutions of a
genomic-DNA-free THP-1 monocytic cell line cDNA stock. A threshold of
detection was set based on the duplicate control samples lacking a
template. Mean sample IL-8 cDNA levels were quantified in THP-1
relative units and normalized to similarly quantified GAPDH cDNA
levels, to control for loading and reverse transcription
efficiencies. Thus, relative IL-8 units were expressed in dimensionless
units, calculated according to the following formula: relative IL-8
units = sample IL-8 units (expressed as equivalent THP-1 IL-8
units)/sample GAPDH units (expressed as equivalent THP-1 GAPDH
units).
Primer/probe sets were as follows: GAPDH forward primer (5'-GCCTTCCGTGTCCCCACTGC-3'), reverse primer (5'-TGAGGGGGCCCTCGACG3'), and probe (5'-tetrachloro-6-carboxyfluorescein-CCTGCTTCACCACCTTCTTGATGTCATCATA-6-carboxytetramethylrhodamine-3'); and IL-8 forward primer (5'-AACATGACTTCCAAGCTGGCCGTG-3'), reverse primer (5'-ACTCCTTGGCAAAACTGCACCTTCAC-3'), and probe (5'-6-carboxyfluorescein-CTCTCTTGGCAGCCTTCCTGATTTCTG-6-carboxytetramethylrhodamine-3').
Statistics
Comparison of two groups was performed using the Student t test, and comparison of more than two data sets was performed using ANOVA and Tukey’s post-test, using the Prism 3.0 program (GraphPad, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocytes consistently expressed the highest levels of TLR2, TLR4, and
CD14 proteins. The staining of neutrophils with all these Abs resulted
in only very small shifts in the FL-2 histograms, but these were
observed in every donor, and mean data were statistically significant.
Eosinophils showed a baseline shift for control Abs when compared with
neutrophils and monocytes, due to their known autofluorescence
(33), and at the protein level were consistently negative
in all donors for expression of TLR2, TLR4, and CD14. In contrast, in
both mixed PBMC populations and in purified cell preparations
(n = 3 for each), basophils consistently expressed low
levels of TLR2 and TLR4, but no CD14 protein. In comparison, we
examined TLR and CD14 expression on neutrophils, eosinophils, and
monocytes in whole blood (Fig. 2). Once
again, eosinophils showed no detectable TLR or CD14 expression.
Monocytes expressed TLR2, TLR4, and CD14 in whole blood with an
identical pattern to that seen in purified PBMC populations, although
with lower mean fluorescences than were seen in purified cells.
Calculating monocyte TLR fluorescence as a percentage of CD14 levels
(Fig. 2F), we found that the ratios of TLR2:CD14 and
TLR4:CD14 were identical between monocytes stained in whole blood and
monocytes stained in purified PBMC preparations, suggesting that the
lower fluorescences in whole blood represented reduced
sensitivity rather than up-regulation of surface markers during
purification. Consistent with this, neutrophils stained in whole blood
showed very low but significant levels of surface TLR2 and CD14
staining, but no detectable TLR4.
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To correlate patterns of TLR expression with leukocyte LPS responses,
leukocytes were purified by preparative techniques resulting in
nonactivated, LPS-responsive cells (31, 32, 33). Purification
of granulocytes by plasma/Percoll gradients resulted in preparations
typically containing >97% granulocytes, with a mean
monocyte-neutrophil ratio of 1:526 (Table I). Pilot experiments showed that 2% FCS
was required for LPS responsiveness of neutrophils in accordance with
published data (1). Fig. 3, A–D, shows that neutrophils in these populations
responded to stimulation with either LPS or fMLP by shedding L-selectin
and up-regulating CD11b. The presence of a fixed concentration of 0.1
nM fMLP together with the varying concentrations of LPS resulted in
additive modulation of cell surface markers. In additional experiments,
granulocyte preparations were depleted of monocytes by CD14 negative
selection, using a CD14 Ab (MEM-15) that does not block LPS-induced
responses (Ref. 39 and data not shown). Although
neutrophils express low levels of CD14, this was not sufficient to
cause their retention in the negative selection column. The resulting
populations showed a significant reduction in monocyte contamination
with a monocyte-neutrophil ratio of 
1:3000 (Table I). CD14-depleted
neutrophils retained their basal levels of L-selectin and their
responsiveness to fMLP (Fig. 3, E and G).
However, LPS was an order of magnitude less potent at inducing shedding
of L-selectin (Fig. 3F) and was less efficacious in the
up-regulation of CD11b in monocyte-depleted neutrophils compared with
nondepleted cells (Fig. 3G).
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shows that
eosinophils in granulocyte preparations, or when highly purified by
negative magnetic selection (typically >97% pure and at least 92%
pure (Table I
)), shed L-selectin in response to fMLP and IL-5 but not
in response to LPS, whether alone or combined with a fixed low (0.1 nM)
concentration of fMLP. In mixed cell suspensions and in keeping with
published data (40), the eosinophil-stimulating chemokine
eotaxin failed to significantly modulate eosinophil L-selectin
expression but was effective in purified cells.
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A shows
that basophils in mixed PBMC populations responded to stimulation with
fMLP and IL-3 by up-regulating CD11b expression, but basophils in these
cell preparations did not respond to LPS (Fig. 5B).
Purification of basophils by negative magnetic selection resulted in a
single population of cells as judged by FSC/SSC plots in flow
cytometry, and gating of this population showed cells with 97.2%
(±0.37 SEM, n = 5) positive staining for CD123, the
IL-3R. These cells showed minimal L-selectin shedding and marked CD11b
up-regulation in response to IL-3 and fMLP (Fig. 5, C and
E). LPS alone did not induce either of these responses (Fig. 5, D and F). Stimulation of purified basophils
with a combination of 0.1 nM fMLP and either 100 or 1000 ng/ml LPS
resulted in small changes in CD11b expression not seen with either
agonist alone; however, these differences failed to reach statistical
significance.
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B
response elements and which has been exploited in transfection-based
assays as a highly sensitive readout of LPS-induced cell activation
(14, 44). Therefore, we used quantitative real-time PCR to
investigate IL-8 mRNA generation in response to LPS in the populations
of neutrophils and purified eosinophils, in parallel samples from those
experiments investigating changes in L-selectin/CD11b expression.
Purified basophils were also included in these analyses but yielded
insufficient mRNA for quantification of IL-8 levels. Fig. 6 shows that neutrophils, both in
standard cell preparations and in those from which monocytes have been
removed, up-regulated IL-8 mRNA expression in response to LPS.
Eosinophil preparations also showed an apparently similar fold increase
in IL-8 mRNA levels in response to LPS stimulation, but on a background
of 100 times lower IL-8:GAPDH mRNA levels (Fig. 6).
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, A–D, shows that
culture in the absence of added stimuli induced up-regulation of TLR2
expression at 4 and 22 h on neutrophils (with no differences
observed between nonapoptotic and apoptotic cells in TLR2 expression at
these time points; data not shown), without any change in TLR4
expression. Coculture with LPS at 100 or 1000 ng/ml prevented the
up-regulation of TLR2 expression seen after 4 h of culture in
every experiment, as illustrated in Fig. 7B (although these
data failed to achieve significance when expressed as changes in mean
fluorescence; Fig. 7E), but did not affect the TLR2
up-regulation seen at 22 h. TLR4 expression was unaffected by
culture in any condition for any time point tested (Fig. 7D
and data not shown).
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A). However, in the 22-h
cultures, LPS alone had no significant effect in preserving cell
viability and protecting from apoptosis as measured by ToPro-3 staining
and cytospin morphology, respectively. In parallel samples, we added
low numbers of monocytes by coincubation of neutrophils with autologous
PBMC populations at 1.5 x 106 or 1.5
x 105 cells/ml (monocytes typically constitute
10% of circulating PBMC; thus, these cocultures were performed in
the presence of 1.5 x 105 or 1.5 x
104 monocytes/ml). By flow cytometry FSC/SSC
plots, the lower concentration of added PBMC corresponded to a mean
monocyte-neutrophil ratio of 1:125 (range, 1:68–226). In the absence
of exogenous stimuli, added monocytes at low or high concentration did
not affect neutrophil survival, but, in the presence of LPS (100
ng/ml), neutrophil apoptosis was almost completely abolished and
viability was preserved to similar levels as seen at time 0. To confirm
that these effects were not mediated through depletion of a strongly
CD14+ neutrophil subset, we purified neutrophils
using a custom mixture that depleted monocytes by anti-CD36 Abs.
These neutrophils responded to LPS (both commercial and repurified) in
assays of L-selectin shedding and CD11b up-regulation, and showed
reduced apoptosis in response to LPS at 4 h but not 22 h, in
keeping with the results above (n = 3; data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils in standard gradient-purified preparations responded to LPS
in a serum-dependent manner, with rapid shedding of L-selectin,
up-regulation of CD11b, and induction of IL-8 mRNA generation, in
keeping with data published by other groups (1, 18, 19, 41). In additional experiments, we depleted neutrophils of
residual monocytes by CD14 negative selection (using a mAb that did not
affect CD14 function). This resulted in a decrease in numbers of cells
in an estimated monocyte FSC/SSC gate, which, although containing only
few events, correlated with PBMC numbers as measured by cytospins. We
observed that these cells continued to make IL-8 mRNA in response to
LPS stimulation, but, in contrast, responses to LPS as measured by
L-selectin shedding and CD11b up-regulation were significantly reduced
(while responses to fMLP were unimpaired). These data suggest that
neutrophil signaling in response to LPS was unimpaired, but that the
adhesion molecule changes were amplified by the presence of monocytes,
probably through the LPS-induced secretion of other monocyte-derived
proinflammatory mediators. To investigate neutrophil LPS responses in
more detail, we studied the regulation of life span in CD14-depleted
neutrophil preparations, and in some samples added back monocytes at
two different concentrations. In monocyte-depleted neutrophil
preparations, LPS prevented the low levels of apoptosis seen after
culture for 4 h. Culture of these cells for 22 h in medium
alone resulted in a population of which most showed apoptotic
morphology and approximately one-third had become nonviable. In
contrast to the findings at 4 h, the presence of LPS throughout
the 22-h culture did not significantly affect neutrophil apoptosis
rates or viability, nor did addition of monocytes (mean
monocyte:neutrophil ratio, 1:125 at low density) when cultured in
medium alone. Strikingly, when LPS was present in the
neutrophil/monocyte coculture, there was an almost complete abrogation
of neutrophil apoptosis and preservation of cell viability. The ability
of LPS-stimulated monocytes to promote neutrophil survival is not in
itself surprising, because stimulated monocytes make survival factors
such as IL-1 and GM-CSF (24, 25). However, the failure of
LPS to prolong neutrophil survival in the 22-h cultures in the absence
of added monocytes suggests that previously observed responses of
neutrophil to LPS may in part have been dependent upon low levels of
monocyte contamination (typical contamination levels of 1–3% PBMC in
isolated neutrophil preparations would result in a final proportion of
0.1–0.3% monocytes). We also subsequently studied neutrophils that
had been depleted of monocytes using a non-CD14-selecting Ab mixture,
and found again that these cells showed reduced apoptosis in response
to LPS at 4 h, but not at 22 h, demonstrating that the
results above were not due to the CD14-mediated depletion of a more
responsive neutrophil subset. Previously, enhanced neutrophil survival
following LPS stimulation has been attributed to autocrine IL-1
release, although these experiments were performed without complete
removal of monocytes (45). Our data show that neutrophils
can respond to LPS with IL-8 mRNA generation and delayed apoptosis at
early time points, but this antiapoptotic effect is lost at later time
points where neutrophils become dependent upon other cells for survival
factors.
Neutrophil TLR/CD14 expression patterns were consistent with the observed LPS responses. Patterns of TLR/CD14 expression for neutrophils, eosinophils, and monocytes were similar in whole blood and purified cells, suggesting that the preparative techniques used had not resulted in modulation of TLR expression, and although we cannot exclude the possibility that cell preparation modified LPS responsiveness it appears to be relatively unlikely. Interestingly, in cultured neutrophils the expression of TLR2, but not TLR4, was regulated. TLR2 expression was up-regulated in cultured neutrophils, an effect prevented by coincubation with LPS at early time points. Our data are in keeping with a previous study that showed TLR2 expression on neutrophils was down-regulated by LPS exposure at early but not late time points (46), although, in contrast to our data, this study found that prolonged (20-h) culture in medium alone caused a small decrease in TLR2 expression. These differences between our data and those of Flo et al. (46) are unlikely to be due to the lack of monocyte depletion in the latter study, as when we added monocytes back to the culture the basal expression of TLR2 was not altered. Furthermore, at the mRNA level in polymorphonuclear leukocytes expression of both TLR2 and TLR4 is up-regulated within 3 h of LPS stimulation (47), although we observed changes only in protein expression in TLR2, suggesting that regulation of TLR2 (46) and TLR4 expression at the protein and mRNA levels is different and potentially complex. In monocytes and macrophages, stimulation by LPS or related ligands results in TLR2 and/or TLR4 mRNA up-regulation (47, 48, 49, 50). One recent study described up-regulation of functional TLR4 by LPS in monocytic-differentiated HL-60 cells but not in granulocytic-differentiated cells (51), and Flo et al. (46) showed that regulation of TLR2 protein expression by cytokines and LPS was different between granulocytes and monocytes, demonstrating lineage-specific patterns of regulation of expression and function.
TLR4 is the major receptor involved in responses to the majority of LPS species (7), with TLR2 involved predominantly in responses to bacterial lipopeptides, including those contained in commercial LPS preparations such as those we used in this study (10, 14, 52, 53, 54). Because TLR2 is expressed at apparently higher levels on neutrophils than is TLR4, and because its expression is regulated by culture and LPS exposure while that of TLR4 is not, it would be tempting to speculate that TLR2 is the dominant receptor on neutrophils accounting for responses to commercial LPS preparations through lipopeptide contaminants. We have also found that the selective TLR2 ligand synthetic bacterial lipopeptide can induce L-selectin shedding and CD11b up-regulation in purified neutrophils (data not shown). However, like the IL-1R (55), TLR4 is functional at extremely low receptor copy number (a few hundred receptors per cell in immature dendritic cells (56)), and we have found that repurified LPS, which signals exclusively via TLR4 (10), is a potent stimulator of neutrophil CD11b and L-selectin responses (efficacious at <10 ng/ml) and an effective inhibitor of neutrophil apoptosis after 4 h of culture, showing that the low levels of TLR4 on neutrophils are functional (data not shown).
Our study is the first to examine eosinophil TLR protein expression, and we found that peripheral blood eosinophils did not express TLR2, TLR4, or CD14, although at the mRNA level there was evidence for TLR4 expression. These data add to recent but conflicting reports of the LPS responsiveness of eosinophils. One of these studies showed that eosinophils expressed TLR2 and TLR4 mRNA and low levels of CD14 protein, and demonstrated LPS responsiveness in assays of cytokine generation (26). However, this study did not deplete eosinophil populations of CD14+ cells. A second study examined eosinophil apoptosis rates in response to LPS and found, by comparing CD14-depleted and non-CD14-depleted cell populations (analogous to our data obtained in neutrophils above), that prevention of eosinophil apoptosis by LPS was monocyte dependent (27). This latter group also showed that eosinophils did not express CD14. We found no evidence of eosinophil LPS responsiveness in assays of L-selectin shedding and CD11b up-regulation, whether in mixed populations or in those that had been purified by a mixture of mAbs including CD14 depletion with a nonblocking CD14 mAb. In the purified eosinophil preparations we observed very low levels of IL-8 mRNA generation in response to LPS, but these were at levels consistent with the very low neutrophil contamination of the purified eosinophil preparations, suggesting that the IL-8 mRNA response probably originated from neutrophils. Thus, our data are in agreement with Meerschaert et al. (27), and we find no evidence that peripheral blood eosinophils are responsive to LPS.
Our data suggest a similar story for the basophil. Only a few studies have examined the responses of basophils to LPS, most of which have shown that in mixed cell suspensions LPS primes or enhances basophil histamine release (20, 28). However, one study showed that purified basophils did not make IL-8 mRNA when stimulated with LPS, but did make IL-8 mRNA in response to control stimuli (43). L-selectin shedding and CD11b up-regulation are inducible in the basophil and correlate with histamine release induced by a variety of secretagogues (57). We found that circulating basophils showed no response to LPS in these assays, although control stimuli were effective. At the protein level, basophils did not express all the components of the LPS receptor, because they expressed TLR2 and TLR4 at levels similar to or greater than those in neutrophils, but not CD14. However, lack of membrane CD14 is not necessarily a bar to LPS responsiveness (58), and soluble CD14 is present in FCS as used in all our assays (59), which has been shown to enable LPS responses in some, but not all, CD14-negative cells (60, 61). Soluble CD14 is effectively delivered to sites of allergic inflammation (62), and the presence of TLR2 and TLR4 on the basophil suggests that LPS responsiveness in this cell type may be inducible at sites of inflammation. Due to a lack of reagents, we were unable to investigate expression of the LPS coreceptor MD-2 in basophils, but it is also possible that a lack of MD-2 contributes to their nonresponsiveness to LPS.
Signaling via TLRs may modulate many aspects of inflammatory responses. TLR4 signals in response to the endogenous proteins heat shock protein-60 and fibrinogen (63, 64) and to exogenous nonbacterial stimuli such as respiratory syncytial virus (65). Allergic inflammatory responses may be significantly modified by infective stimuli (15, 16, 66, 67). Our data suggest that the monocyte is a key orchestrator of LPS responsiveness and that, even for neutrophils, its contribution to observed LPS responses is highly significant.
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Moira K. B. Whyte, Division of Genomic Medicine, University of Sheffield, M Floor, Royal Hallamshire Hospital, Sheffield S10 2JF, U.K. E-mail address: m.k.whyte{at}sheffield.ac.uk
3 Abbreviations used in this paper: TLR, Toll-like receptor; FSC, forward light scatter; SSC, side light scatter; RT, reverse transcriptase.
Received for publication November 26, 2001. Accepted for publication February 20, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and IL-8 release by human monocytes and alveolar macrophages. J. Immunol. 150:2885.[Abstract]
-converting enzyme (caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1. J. Immunol. 161:957.This article has been cited by other articles:
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  V. Driss, F. Legrand, E. Hermann, S. Loiseau, Y. Guerardel, L. Kremer, E. Adam, G. Woerly, D. Dombrowicz, and M. Capron TLR2-dependent eosinophil interactions with mycobacteria: role of {alpha}-defensins Blood, April 2, 2009; 113(14): 3235 - 3244. [Abstract] [Full Text] [PDF]
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 Y.-L. Guo, R. Bai, C. X-J Chen, D.-Q. Liu, Y. Liu, C.-Y. Zhang, and K. Zen Role of Junctional Adhesion Molecule-Like Protein in Mediating Monocyte Transendothelial Migration Arterioscler. Thromb. Vasc. Biol., January 1, 2009; 29(1): 75 - 83. [Abstract] [Full Text] [PDF]
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 V. Stadlbauer, R. P. Mookerjee, G. A. K. Wright, N. A. Davies, G. Jurgens, S. Hallstrom, and R. Jalan Role of Toll-like receptors 2, 4, and 9 in mediating neutrophil dysfunction in alcoholic hepatitis Am J Physiol Gastrointest Liver Physiol, January 1, 2009; 296(1): G15 - G22. [Abstract] [Full Text] [PDF]
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 L. C. Parker, E. C. Prestwich, J. R. Ward, E. Smythe, A. Berry, M. Triantafilou, K. Triantafilou, and I. Sabroe A Phosphatidylserine Species Inhibits a Range of TLR- but Not IL-1{beta}-Induced Inflammatory Responses by Disruption of Membrane Microdomains J. Immunol., October 15, 2008; 181(8): 5606 - 5617. [Abstract] [Full Text] [PDF]
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 E. P. Dick, L. R. Prince, and I. Sabroe Ex Vivo-Expanded Bone Marrow CD34+ Derived Neutrophils Have Limited Bactericidal Ability Stem Cells, October 1, 2008; 26(10): 2552 - 2563. [Abstract] [Full Text] [PDF]
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B. Koller, M. Kappler, P. Latzin, A. Gaggar, M. Schreiner, S. Takyar, M. Kormann, M. Kabesch, D. Roos, M. Griese, et al. TLR Expression on Neutrophils at the Pulmonary Site of Infection: TLR1/TLR2-Mediated Up-Regulation of TLR5 Expression in Cystic Fibrosis Lung Disease J. Immunol., August 15, 2008; 181(4): 2753 - 2763. [Abstract] [Full Text] [PDF]
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 C.-S. Lin, J. G. Wann, C.-W. Hsiao, D. W. Tai, J.-S. Chen, Y.-H. Hsu, H.-C. Huang, and C.-F. Chao Intracellular acidification enhances neutrophil phagocytosis in chronic haemodialysis patients: possible role of CD11b/CD18 Nephrol. Dial. Transplant., May 1, 2008; 23(5): 1642 - 1649. [Abstract] [Full Text] [PDF]
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 P. F.-Y. Cheung, C.-K. Wong, W.-K. Ip, and C. W.-K. Lam FAK-mediated activation of ERK for eosinophil migration: a novel mechanism for infection-induced allergic inflammation Int. Immunol., March 1, 2008; 20(3): 353 - 363. [Abstract] [Full Text] [PDF]
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L. R. Prince, S. M. Bianchi, K. M. Vaughan, M. A. Bewley, H. M. Marriott, S. R. Walmsley, G. W. Taylor, D. J. Buttle, I. Sabroe, D. H. Dockrell, et al. Subversion of a Lysosomal Pathway Regulating Neutrophil Apoptosis by a Major Bacterial Toxin, Pyocyanin J. Immunol., March 1, 2008; 180(5): 3502 - 3511. [Abstract] [Full Text] [PDF]
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 K. P. Mollen, R. M. Levy, J. M. Prince, R. A. Hoffman, M. J. Scott, D. J. Kaczorowski, R. Vallabhaneni, Y. Vodovotz, and T. R. Billiar Systemic inflammation and end organ damage following trauma involves functional TLR4 signaling in both bone marrow-derived cells and parenchymal cells J. Leukoc. Biol., January 1, 2008; 83(1): 80 - 88. [Abstract] [Full Text] [PDF]
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K. R. Vaughan, L. Stokes, L. R. Prince, H. M. Marriott, S. Meis, M. U. Kassack, C. D. Bingle, I. Sabroe, A. Surprenant, and M. K. B. Whyte Inhibition of Neutrophil Apoptosis by ATP Is Mediated by the P2Y11 Receptor J. Immunol., December 15, 2007; 179(12): 8544 - 8553. [Abstract] [Full Text] [PDF]
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 I. Gillette-Ferguson, K. Daehnel, A. G. Hise, Y. Sun, E. Carlson, E. Diaconu, H. F. McGarry, M. J. Taylor, and E. Pearlman Toll-Like Receptor 2 Regulates CXC Chemokine Production and Neutrophil Recruitment to the Cornea in Onchocerca volvulus/ Wolbachia-Induced Keratitis Infect. Immun., December 1, 2007; 75(12): 5908 - 5915. [Abstract] [Full Text] [PDF]
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 C. K. Wong, P. F. Y. Cheung, W. K. Ip, and C. W. K. Lam Intracellular Signaling Mechanisms Regulating Toll-Like Receptor-Mediated Activation of Eosinophils Am. J. Respir. Cell Mol. Biol., July 1, 2007; 37(1): 85 - 96. [Abstract] [Full Text] [PDF]
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H. D'Avila, P. E. Almeida, N. R. Roque, H. C. Castro-Faria-Neto, and P. T. Bozza Toll-Like Receptor-2-Mediated C-C Chemokine Receptor 3 and Eotaxin-Driven Eosinophil Influx Induced by Mycobacterium bovis BCG Pleurisy Infect. Immun., March 1, 2007; 75(3): 1507 - 1511. [Abstract] [Full Text] [PDF]
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M. J. Flagler, J. E. Strasser, C. L. Chalk, and A. A. Weiss Comparative Analysis of the Abilities of Shiga Toxins 1 and 2 To Bind to and Influence Neutrophil Apoptosis Infect. Immun., February 1, 2007; 75(2): 760 - 765. [Abstract] [Full Text] [PDF]
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 G. E. Morris, L. C. Parker, J. R. Ward, E. C. Jones, M. K. B. Whyte, C. E. Brightling, P. Bradding, S. K. Dower, and I. Sabroe Cooperative molecular and cellular networks regulate Toll-like receptor-dependent inflammatory responses FASEB J, October 1, 2006; 20(12): 2153 - 2155. [Abstract] [Full Text] [PDF]
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C. A. Lindemans, P. J. Coffer, I. M. M. Schellens, P. M. A. de Graaff, J. L. L. Kimpen, and L. Koenderman Respiratory Syncytial Virus Inhibits Granulocyte Apoptosis through a Phosphatidylinositol 3-Kinase and NF-{kappa}B-Dependent Mechanism J. Immunol., May 1, 2006; 176(9): 5529 - 5537. [Abstract] [Full Text] [PDF]
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 R. E. Rumbaut, R. V. Bellera, J. K. Randhawa, C. N. Shrimpton, S. K. Dasgupta, J.-F. Dong, and A. R. Burns Endotoxin enhances microvascular thrombosis in mouse cremaster venules via a TLR4-dependent, neutrophil-independent mechanism Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1671 - H1679. [Abstract] [Full Text] [PDF]
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M. Bonneau, M. Epardaud, F. Payot, V. Niborski, M.-I. Thoulouze, F. Bernex, B. Charley, S. Riffault, L. A. Guilloteau, and I. Schwartz-Cornil Migratory monocytes and granulocytes are major lymphatic carriers of Salmonella from tissue to draining lymph node J. Leukoc. Biol., February 1, 2006; 79(2): 268 - 276. [Abstract] [Full Text] [PDF]
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S. A. Tavener and P. Kubes Cellular and molecular mechanisms underlying LPS-associated myocyte impairment Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H800 - H806. [Abstract] [Full Text] [PDF]
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L. C. Parker, E. C. Jones, L. R. Prince, S. K. Dower, M. K. B. Whyte, and I. Sabroe Endotoxin tolerance induces selective alterations in neutrophil function J. Leukoc. Biol., December 1, 2005; 78(6): 1301 - 1305. [Abstract] [Full Text] [PDF]
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 C. Marsik, B. Jilma, C. Joukhadar, C. Mannhalter, O. Wagner, and G. Endler The Toll-Like Receptor 4 Asp299Gly and Thr399Ile Polymorphisms Influence the Late Inflammatory Response in Human Endotoxemia Clin. Chem., November 1, 2005; 51(11): 2178 - 2180. [Full Text] [PDF]
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K. Hattar, S. van Burck, A. Bickenbach, U. Grandel, U. Maus, J. Lohmeyer, E. Csernok, T. Hartung, W. Seeger, F. Grimminger, et al. Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation J. Leukoc. Biol., October 1, 2005; 78(4): 992 - 1000. [Abstract] [Full Text] [PDF]
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G. Andonegui, S. M. Kerfoot, K. McNagny, K. V. J. Ebbert, K. D. Patel, and P. Kubes Platelets express functional Toll-like receptor-4 Blood, October 1, 2005; 106(7): 2417 - 2423. [Abstract] [Full Text] [PDF]
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 T. V. Pedchenko, G. Y. Park, M. Joo, T. S. Blackwell, and J. W. Christman Inducible binding of PU.1 and interacting proteins to the Toll-like receptor 4 promoter during endotoxemia Am J Physiol Lung Cell Mol Physiol, September 1, 2005; 289(3): L429 - L437. [Abstract] [Full Text] [PDF]
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L. C. Parker, M. K. B. Whyte, S. K. Dower, and I. Sabroe The expression and roles of Toll-like receptors in the biology of the human neutrophil J. Leukoc. Biol., June 1, 2005; 77(6): 886 - 892. [Abstract] [Full Text] [PDF]
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 G. E. Morris, M. K. B. Whyte, G. F. Martin, P. J. Jose, S. K. Dower, and I. Sabroe Agonists of Toll-like Receptors 2 and 4 Activate Airway Smooth Muscle via Mononuclear Leukocytes Am. J. Respir. Crit. Care Med., April 15, 2005; 171(8): 814 - 822. [Abstract] [Full Text] [PDF]
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S. Francois, J. El Benna, P. M. C. Dang, E. Pedruzzi, M.-A. Gougerot-Pocidalo, and C. Elbim Inhibition of Neutrophil Apoptosis by TLR Agonists in Whole Blood: Involvement of the Phosphoinositide 3-Kinase/Akt and NF-{kappa}B Signaling Pathways, Leading to Increased Levels of Mcl-1, A1, and Phosphorylated Bad J. Immunol., March 15, 2005; 174(6): 3633 - 3642. [Abstract] [Full Text] [PDF]
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G. Tunheim, K. W. Schjetne, A. B. Fredriksen, I. Sandlie, and B. Bogen Human CD14 is an efficient target for recombinant immunoglobulin vaccine constructs that deliver T cell epitopes J. Leukoc. Biol., March 1, 2005; 77(3): 303 - 310. [Abstract] [Full Text] [PDF]
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U. A. Maus, S. Wellmann, C. Hampl, W. A. Kuziel, M. Srivastava, M. Mack, M. B. Everhart, T. S. Blackwell, J. W. Christman, D. Schlondorff, et al. CCR2-positive monocytes recruited to inflamed lungs downregulate local CCL2 chemokine levels Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L350 - L358. [Abstract] [Full Text] [PDF]
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S. Bellocchio, S. Moretti, K. Perruccio, F. Fallarino, S. Bozza, C. Montagnoli, P. Mosci, G. B. Lipford, L. Pitzurra, and L. Romani TLRs Govern Neutrophil Activity in Aspergillosis J. Immunol., December 15, 2004; 173(12): 7406 - 7415. [Abstract] [Full Text] [PDF]
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 L. R. Prince, L. Allen, E. C. Jones, P. G. Hellewell, S. K. Dower, M. K.B. Whyte, and I. Sabroe The Role of Interleukin-1{beta} in Direct and Toll-Like Receptor 4-Mediated Neutrophil Activation and Survival Am. J. Pathol., November 1, 2004; 165(5): 1819 - 1826. [Abstract] [Full Text] [PDF]
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L. C. Parker, M. K. B. Whyte, S. N. Vogel, S. K. Dower, and I. Sabroe Toll-Like Receptor (TLR)2 and TLR4 Agonists Regulate CCR Expression in Human Monocytic Cells J. Immunol., April 15, 2004; 172(8): 4977 - 4986. [Abstract] [Full Text] [PDF]
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 J. S. Park, D. Svetkauskaite, Q. He, J.-Y. Kim, D. Strassheim, A. Ishizaka, and E. Abraham Involvement of Toll-like Receptors 2 and 4 in Cellular Activation by High Mobility Group Box 1 Protein J. Biol. Chem., February 27, 2004; 279(9): 7370 - 7377. [Abstract] [Full Text] [PDF]
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 E. Eldering, C. A. Spek, H. L. Aberson, A. Grummels, I. A. Derks, A. F. de Vos, C. J. McElgunn, and J. P. Schouten Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways Nucleic Acids Res., December 1, 2003; 31(23): e153 - e153. [Abstract] [Full Text] [PDF]
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H. Nagase, S. Okugawa, Y. Ota, M. Yamaguchi, H. Tomizawa, K. Matsushima, K. Ohta, K. Yamamoto, and K. Hirai Expression and Function of Toll-Like Receptors in Eosinophils: Activation by Toll-Like Receptor 7 Ligand J. Immunol., October 15, 2003; 171(8): 3977 - 3982. [Abstract] [Full Text] [PDF]
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M. Choi, S. Rolle, M. Wellner, M. C. Cardoso, C. Scheidereit, F. C. Luft, and R. Kettritz Inhibition of NF-{kappa}B by a TAT-NEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis Blood, September 15, 2003; 102(6): 2259 - 2267. [Abstract] [Full Text] [PDF]
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I. Sabroe, R. C. Read, M. K. B. Whyte, D. H. Dockrell, S. N. Vogel, and S. K. Dower Toll-Like Receptors in Health and Disease: Complex Questions Remain J. Immunol., August 15, 2003; 171(4): 1630 - 1635. [Full Text] [PDF]
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E. Abraham, M. R. Gyetko, K. Kuhn, J. Arcaroli, D. Strassheim, J. S. Park, S. Shetty, and S. Idell Urokinase-Type Plasminogen Activator Potentiates Lipopolysaccharide-Induced Neutrophil Activation J. Immunol., June 1, 2003; 170(11): 5644 - 5651. [Abstract] [Full Text] [PDF]
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G. J. Nau, A. Schlesinger, J. F. L. Richmond, and R. A. Young Cumulative Toll-Like Receptor Activation in Human Macrophages Treated with Whole Bacteria J. Immunol., May 15, 2003; 170(10): 5203 - 5209. [Abstract] [Full Text] [PDF]
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I. Sabroe, L. R. Prince, E. C. Jones, M. J. Horsburgh, S. J. Foster, S. N. Vogel, S. K. Dower, and M. K. B. Whyte Selective Roles for Toll-Like Receptor (TLR)2 and TLR4 in the Regulation of Neutrophil Activation and Life Span J. Immunol., May 15, 2003; 170(10): 5268 - 5275. [Abstract] [Full Text] [PDF]
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 E. Mattsson, T. Persson, P. Andersson, J. Rollof, and A. Egesten Peptidoglycan Induces Mobilization of the Surface Marker for Activation Marker CD66b in Human Neutrophils but Not in Eosinophils Clin. Vaccine Immunol., May 1, 2003; 10(3): 485 - 488. [Abstract] [Full Text] [PDF]
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A. Ayala, C.-S. Chung, J. L. Lomas, G. Y. Song, L. A. Doughty, S. H. Gregory, W. G. Cioffi, B. W. LeBlanc, J. Reichner, H. H. Simms, et al. Shock-Induced Neutrophil Mediated Priming for Acute Lung Injury in Mice: Divergent Effects of TLR-4 and TLR-4/FasL Deficiency Am. J. Pathol., December 1, 2002; 161(6): 2283 - 2294. [Abstract] [Full Text] [PDF]
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), or varying concentrations of LPS
plus a fixed concentration (0.1 nM) of fMLP (
). After stimulation
they were washed, and L-selectin (A, B,
E, and F) and CD11b (C,
D, G, and H) surface
expression was measured by flow cytometry and quantified as described
in Materials and Methods. Significant
differences in induction of L-selectin shedding and CD11b up-regulation
by LPS between neutrophils in mixed populations and those further
purified by negative selection are indicated: *,
p < 0.05; ***, p <
0.001. Data are shown as mean ± SEM from four to seven
experiments.
), or IL-5 (
). After stimulation they were
washed, and L-selectin (A–D) and CD11b
(E and F) surface expression was measured
by flow cytometry and quantified as described in Materials
and Methods. Data are shown as mean ± SEM
from four to five separate experiments.
