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Department of Pediatrics and the Immunology Program, Stanford University School of Medicine, Stanford, CA 94304
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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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and IL-12, and costimulatory molecules, such
as CD80 (B7-1) and CD86 (B7-2) (2, 6, 7) via a
MyD88/IL-IR-associated kinase/TNFR-associated factor 6/NF-
B
signaling pathway (8, 9) as well as by a poorly
characterized MyD88-independent pathway (6). TLR4 is
expressed at particularly high levels by cells of the innate immune
system, such as mononuclear phagocytes and dendritic cells (Ref.
10 and M. E. Dahl and D. B. Lewis, unpublished
observations).
Dendritic cells are crucial to the initiation of adaptive immune
responses because of their ability to efficiently process protein Ags
and present antigenic peptides on class I and II MHC molecules to
antigenically naive T cells (11). Naive T cell activation
by dendritic cells is also particularly efficient because dendritic
cells express relatively high basal levels of molecules involved in
costimulation, such as CD40, CD80, and CD86 (11, 12).
There is also growing evidence that dendritic cells help direct the
differentiation of naive CD4 T cells into either Th1 or Th2
effector/memory cells capable of producing either IFN-
or IL-4,
IL-5, and IL-13, respectively (12, 13). These findings
have generated a growing interest in defining how microbial products
and their respective receptors on dendritic cells may regulate Th1- vs
Th2-mediated adaptive immunity (12, 14)
Allergen-induced asthma is a disease in which the CD4 Th2 immune response plays a pivotal role and is characterized by high circulating levels of IgE, pulmonary eosinophilic inflammation, and airway hyperreactivity to bronchoconstrictive stimuli (14). Although dendritic cells are likely to play a key role in the skewing of adaptive immune responses toward Th2 responses, the mechanisms by which this occurs remain controversial (15). Since CD86 may play a critical role in the induction of experimental allergen-induced asthma in rodents (16), one plausible mechanism for such skewing may be by the up-regulation of CD86 surface expression on dendritic cells in the absence of other events that promote Th1 skewing, such as the secretion of IL-12.
The role of LPS exposure in the induction of asthma or in modulating
existing disease is also poorly understood, but appears to be complex,
reflecting the timing of exposure as well as the particular parameter
of Th2-mediated pathology. For example, LPS administration to rodents
with experimentally induced asthma prevents the development of Th2
responses and pulmonary inflammation as well as the associated airway
hyperreactivity (17, 18), but may increase IgE production
when given before antigenic sensitization (19). The
inhibitory effects of LPS on Th2 responses are consistent with LPS
inducing IL-12 and IFN- (20), which in some
allergen-induced asthma models block Th2 and favor Th1 responses
(21). Additional human studies have variously reported an
association of LPS exposure with an increased risk of asthma-like
respiratory symptoms (22) or with a decreased risk of
sensitization to aeroallergens (23). A promoter
polymorphism in the human gene encoding CD14 that correlates with
increased levels of CD14 in the circulation (24) and on
mononuclear phagocytes (25) and with decreased circulating
IgE levels (24, 26) has also been interpreted as evidence
of increased LPS signaling having an inhibitory effect on Th2
responses. However, the increased expression of CD14 associated with
this polymorphism may not necessarily mediate its effects by increased
activation of TLR4 by LPS, since CD14 also interacts with ligands for
TLR2 such as peptidoglycan (1).
To address the potential role of TLR4 activation in regulating
Th2-mediated responses, we compared experimental allergen-induced
asthma in C3H/HeJ mice, a strain that lacks functional TLR4 receptors
due to a Pro712His substitution mutation in the
cytoplasmic domain that abolishes intracellular signaling
(27), with those of C3H/HeOuJ mice, which are wild type
(WT) for this receptor but are otherwise similar in genetic background
(Ref. 27 and references therein). We predicted that
a lack of TLR4 signaling in C3H/H3J mice, hereafter referred to as
TLR4-defective mice, would impair Th1-type immune responses as a result
of reduced production of IL-12 and IFN-, thereby favoring Th2-type
responses. Surprisingly, TLR4-defective mice were found to have
markedly reduced Th2 immune responses to allergen, and that this was
attributable, at least in part, to diminished dendritic cell
function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/HeJ (TLR4 defective), C3H/HeOuJ (WT control), and B10.A mice
(all of the H-2k haplotype) were purchased from
The Jackson Laboratory (Bar Harbor, ME). AND transgenic mice, in which
>85% of CD4 T cells bear a transgene-encoded 
-TCR reactive with
an I-Ek-restricted peptide of pigeon cytochrome
c protein (PCC) (28), were obtained from Dr. S.
Hedrick (University of California, San Diego, CA) and maintained on a
B10.A background. All animals were kept under strict specific
pathogen-free conditions at the Stanford University Research Animal
Facility, and all procedures were in accordance with Stanford
University guidelines.
Immunization and bronchoalveolar lavage (BAL) fluid cell collection
Eight- to 12-wk-old female TLR4-defective or WT control mice were immunized with 100 µg of crystalline and LPS-free OVA (Pierce, Rockford, IL) in alum or with alum alone by i.p injection on days 1 and 14 as previously described (29). Alum (aluminum hydroxide hydrate) powder was purchased (Sigma-Aldrich, St. Louis, MO) and reconstituted in LPS-free sterile water for irrigation (Baxter Healthcare, Deerfield, IL). LPS was undetectable in the 10% alum solution used for preparation of OVA injections, based on the E-Toxate Limulus lysate test (Sigma-Aldrich), which has a lower limit of sensitivity (0.05–0.1 endotoxin units/ml). OVA (100 µg) in PBS or PBS alone was administered intranasally (i.n.) after light anesthesia on days 14, 24, and 25, as described previously (29). BAL fluid was obtained following euthanasia of mice on day 26 using 1.2 ml of PBS, 0.1% BSA, and 0.5 mM EDTA, and total cell counts were determined (29). Microscope slides of cells obtained by BAL were prepared by cytocentrifugation and were stained with Diff-Quik (Fisher, Pittsburgh, PA) (30). Differential cell counts were performed by counting at least 300 cells per slide.
OVA-specific IgE and IgG1 ELISAs
OVA-specific IgG1 was determined as previously described (31). To measure OVA-specific IgE, wells of Maxisorp (Nunc, Roskilde, Denmark), ELISA plates were coated with goat anti-mouse IgE (Southern Biotechnology Associates, Birmingham, AL) in bicarbonate buffer at pH 9.5 at 4°C overnight and washed with PBS with 0.05% Tween 20 (ELISA buffer). All subsequent steps were performed at 37°C with extensive washing between steps. Plates were blocked with PBS with 3% FCS, and plasma samples diluted (1/25) in ELISA buffer were added to the wells for 2 h. This was followed by a 1-h incubation with biotinylated OVA (1/500 dilution) of a stock solution previously generated using a biotinylation kit (Sigma-Aldrich) following the manufacturer’s instructions. Wells were incubated with streptavidin-conjugated peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at 37°C, developed using tetramethylbenzidine substrate (Kirkegaard & Perry, Gaithersburg, MD), and absorbance at 650 nm was determined using an ELISA plate reader.
OVA-specific T cell responses in vitro
Bronchial lymph node cells were isolated from immunized mice and
cultured (5 x 105/well) as previously
described (30) with medium alone or with OVA (100 mg) for
96 h. IL-4, IL-5, and IFN- content in cell culture supernatants
were determined by ELISA (BD PharMingen, San Diego, CA). Some cultures
were pulsed with [3H]thymidine (1 µCi/well)
for the last 18 h of the incubation period and cell proliferation
was determined by measuring radioactivity incorporated into DNA using
liquid scintillation counting on a Betaplate beta-emitter detection
system (Perkin-Elmer Wallac, Boston, MA).
Dendritic cell purification and flow cytometric analysis
Dendritic cells were isolated from splenic, lymph node, and lung
tissue of mice by mincing tissue, followed by treatment with 300 U/ml
type I collagenase (Worthington Biochemical, Lakewood, NJ) and 100 U/ml
DNase I (Sigma-Aldrich) for 90 min in RPMI 1640 medium without
serum. After filtration through nylon gauze to remove debris, the cells
were resuspended in RPMI 1640 medium with 10% FCS and incubated with
CD11c mAb-coated paramagnetic microbeads and applied to an AutoMacs
according to the manufacturer’s instructions (Miltenyi Biotec, Auburn,
CA). The positively selected cells used for experiments were routinely
>85% CD11c+. This cell fraction was stained
with PE-conjugated CD11c, FITC-conjugated class II MHC (clone 11-5.2
reactive with I-Ak), and either biotinylated
CD40, CD80, or CD86 mAbs (all from BD PharMingen). After washing, cells
were treated with streptavidin-PE-Cy5 (Tricolor; Caltag Laboratories,
Burlingame, CA) and analyzed by three-color flow cytometry using a
FACSCalibur instrument (BD Biosciences, San Jose, CA). Costimulatory
molecule surface expression was then analyzed on immature (class II
MHClow) or mature (class II
MHChigh) CD11c+ dendritic
cells (see Fig. 2
A for gating criteria). PCC-reactive CD4 T
cells were purified from combined splenic and lymph node cell
suspensions of AND transgenic mice by MACS using CD4 mAb-coated
microbeads (Miltenyi Biotec) and the AutoMacs instrument. All cell
cultures were maintained in RPMI 1640 medium supplemented with 10%
FCS, L-glutamine, 50 µg/ml penicillin and
streptomycin, and 2-ME (1 x 10-5 M) at
37°C in a 5% CO2 humidified environment.
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Dendritic cells (1 x 104/well) were
incubated in 96-well round bottom plates with PCC-reactive CD4 T cells
(1 x 105/well) with or without 1 µM PCC
protein (Sigma-Aldrich). CD69 and CD154 (CD40 ligand) surface
expression were assessed at 12 h by staining cells with
FITC-conjugated CD4 (Caltag Laboratories) and either biotin-conjugated
CD154 or PE-conjugated CD69 mAbs (both from BD PharMingen). Cells
stained with CD154 mAb were then washed and incubated with
streptavidin-PE-Cy5. Cell proliferation was measured by pulsing cell
cultures at 24 h with [3H]thymidine (1
µCi/well) and determining incorporation after an additional 24 h
of incubation using a Betaplate apparatus. Some cultures were harvested
at 48 h and IL-4, IL-5, and IFN- levels were measured by ELISA.
To assay for activation-induced CD86 surface expression, dendritic
cells (5 x 105 cells/well) were cultured in
the presence of 10 ng/ml GM-CSF (PeproTech, Rocky Hill, NJ) with or
without 5 µg/ml hamster anti-mouse CD40 mAb (clone 3/23; BD
PharMingen). In cultures treated with CD40 mAb, this was followed by
the addition of goat anti-hamster IgG (5 µg/ml; BD PharMingen)
for 24 h for analysis of CD86 surface expression. Results are only
shown for mature dendritic cells, as stimulation resulted in >95% of
cells expressing high levels of class II MHC (data not shown). IL-12
production after 48 h was determined similarly, except that GM-CSF
was omitted. IL-12 content in cell culture supernatants was determined
using a commercial kit (BD PharMingen) specific for the p40 subunit.
For long-term priming, cultures were incubated for 10–14 days, with
the addition of fresh medium without cytokines every 3 days. Cells were
then harvested by extensive washing and centrifugation, and incubated
in 96-well round bottom plates (1 x 105
cells/well) with an equivalent number of freshly isolated
H-2k-matched splenocytes from B10.A mice and with
or without PCC protein (1 µM). Cell culture supernatants were
collected after 48 h of incubation and analyzed for levels of
IL-4, IL-5, and IFN- by ELISA.
Dendritic cell functional analysis in vivo
CD11c+ dendritic cells were pulsed with or without 1 µM PCC protein for 12 h, extensively washed, and transferred i.n. to anesthetized AND transgenic mice (5 x 105 cells in 50 µl of PBS/animal). After 48 h, BAL of the lungs was performed, and total and differential cell counts were performed as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the development of Th2-type responses in the
absence of TLR4 signals, we sensitized TLR4-defective and WT control
mice using alum-precipitated OVA and challenged these animals with
repeated doses of OVA administered by the i.n. route. TLR4-defective
mice had an overall decrease in lung inflammatory responses compared
with WT mice based on the total number of leukocytes present in BAL
fluid (Fig. 1A). There was a
dramatic and significant reduction in the numbers of eosinophils and
lymphocytes, but no significant differences between TLR4-defective and
WT mice in the number of mononuclear phagocytes or polymorphonuclear
leukocytes. The reduced inflammation with eosinophils, which in this
model is dependent on the production of Th2 cytokine IL-5
(32), suggested a reduction in Th2 responses.
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B), an Ig isotype whose expression is dependent on the
production of the Th2 cytokine IL-4 (31). In contrast,
there were no significant differences between the two groups in the
levels of OVA-specific IgG1 (Fig. 1B), an isotype that is
expressed in substantial amounts in the complete absence of IL-4
(33) or IgG2a (data not shown), an isotype that is
positively regulated by the signature Th1 cytokine IFN-
(34). This suggested that TLR4-defective mice were not
generally compromised in their ability to produce Abs to T-dependent
Ags. Importantly, when lung-draining lymph node cells were cultured in
the presence of OVA in vitro, cells from TLR4-defective mice produced
significantly lower amounts of IL-4 and IL-5 (>95% reduction for both
cytokines) compared with WT mice (Fig. 1C). There was also a
slight but significant reduction in IFN- production by cells from
TLR4-defective mice. Together, these results suggest that
allergen-induced Th2 responses were compromised in the absence of
signaling via TLR4, whereas Th1 responses to allergen were relatively
preserved. Finally, TLR4-defective and WT lymph node cells proliferated
similarly in response to OVA stimulation (data not shown), indicating
that a lack of TLR4 signaling did not generally compromise the in vivo
generation of allergen-specific CD4 T cells. Decreased costimulatory molecule expression and cytokine production by dendritic cells of TLR4-defective mice
We examined whether the absence of TLR4 signaling affected the
number or surface phenotype of splenic, lymph node, and lung
CD11c+ dendritic cells, as such alterations might
influence the outcome of CD4 T cell differentiation (12, 13, 15). There were no differences between TLR4-defective and WT
mice in the numbers of CD11c+ cells isolated from
the spleen, lymph nodes, or lungs (data not shown). Furthermore, there
were no significant differences between WT and TLR-4-defective mice in
the frequency of immature and mature CD11c+ cells
present in the spleen based on MHC class II surface expression (Fig. 2A), the basal cell surface
expression of the costimulatory molecule CD86 (Fig. 2B,
left panels) or of CD40 (data not shown). However,
TLR4-defective dendritic cells expressed significantly less CD86 after
their stimulation with GM-CSF alone or in combination with an
activating CD40 mAb (Fig. 2B, right panels).
Similar reductions were also noted for CD80 expression (data not
shown). Finally, TLR4-defective dendritic cells stimulated with CD40
mAb produced significantly less IL-12 compared with WT cells (Fig. 2C). Thus, the absence of TLR4 signaling in vivo not only
reduced IL-12 production by dendritic cells in response to known TLR4
activators, such as LPS (6), but also for other stimuli
that act independently of TLR4.
Decreased function of TLR4-defective dendritic cells in activating naive CD4 T cells for Th2 cytokine production
To investigate the functional differences that a lack of TLR4
signaling had on dendritic cell function, we studied the in vitro
ability of CD11c+ dendritic cells pulsed with a
protein Ag, PCC, to activate antigenically naive CD4 T cells. CD4 T
cells expressing a -TCR transgene that confers reactivity with a
PCC peptide (28) were used as a homogeneous responder
population. There were no significant differences in CD4 T cell
proliferation (Fig. 3A) or
surface expression of CD154 (CD40 ligand) (Fig. 3B) induced
by TLR4-defective or WT dendritic cells pulsed with PCC protein. CD4 T
cells incubated for 48 h with Ag-pulsed TLR4-defective or WT
dendritic cells also produced similar levels of IFN- and
undetectable levels of IL-4 or IL-5 (data not shown).
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C). Together, these
results suggested that although a lack of TLR4 signaling did not
compromise the initial activation of naive CD4 T cells, it reduced the
priming for Th2 cytokine expression by the effector CD4 T cell
population. Decreased function mediated by TLR4-defective dendritic cells in vivo
To determine whether limitations in TLR4 activation altered the
function of dendritic cells in vivo, PCC-pulsed dendritic cells from
TLR4-defective and WT mice were administered into the lungs of AND
transgenic mice, and pulmonary inflammation was assessed 48 h
later. WT dendritic cells pulsed with PCC protein induced infiltration
of a significantly greater number of lymphocytes and polymorphonuclear
neutrophils in recipient mice compared with dendritic cells from
TLR4-defective mice (Fig. 4). After
pulsing with PCC, WT dendritic cells also tended to induce higher
levels of eosinophils than did those of TLR4-defective mice, although
this difference did not achieve statistical significance. The induction
of these increased numbers of cells required PCC protein pulsing and
was not observed when this was omitted or an irrelevant protein was
used in place of PCC (data not shown). These results suggested that a
lack of TLR4 signaling had a significant effect on the capacity of
dendritic cells to generate a local inflammatory response in vivo in
response to Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The impaired Th2 response of mice defective in TLR4 signaling was accompanied by decreased CD86 expression by dendritic cells in response to GM-CSF treatment alone or in combination with CD40 engagement. CD86 binding to CD28 on T cells has previously been reported to be necessary for the generation of IL-4-producing cells from antigenically naive CD4 T cells (41) and for the induction of Th2-type allergic responses in vivo (42, 43). Therefore, it is likely that decreased expression of this molecule by dendritic cells contributes to the impaired Th2 response of TLR4-defective mice. Importantly, dendritic cells from TLR4-defective mice had a decreased ability compared with those of WT mice for promoting the differentiation of antigenically naive WT CD4 T cells into Th2-type effector cells, as assessed by Ag-specific IL-4 and IL-5 production in vitro. These cells were also less effective in eliciting a CD4 T cell Ag-specific pulmonary inflammatory response than WT dendritic cells in vivo. Because of the short life span of dendritic cells after adoptive transfer, this approach does not allow an evaluation of Th2-mediated inflammation directed by dendritic cells, since this requires repetitive T cell activation to induce Th2 differentiation. Despite these limitations, these adoptive transfer studies suggested that dendritic cells from TLR4-defective mice had a reduced capacity to induce inflammation in response to specific Ag than cells from WT mice. Together, these results strongly suggest that a lack of TLR4 signaling in vivo limited the activation of naive CD4 T cells into effector cells as a consequence of immature or altered dendritic cell function.
In contrast to these effects on Th2-mediated responses, there were no significant differences between TLR4-defective and WT mice for the numbers of dendritic cells found in the spleen, lymph nodes, or lung, the proportion of dendritic cells that were immature (class II MHClow) vs mature (class II MHChigh), or the basal expression of CD40, CD80, and CD86 on either immature or mature dendritic cells. Dendritic cells from mice lacking TLR4 signaling also appeared similar to those of WT mice in their ability to internalize protein and present Ag via the class II MHC pathway, and to activate naive CD4 T cells, as assessed by proliferation and the T cell surface expression of CD69 and CD154. Thus, the absence of TLR4 activation in vivo did not result in any demonstrable quantitative deficiencies in dendritic cells, at least in these tissues, and did not generally arrest the process of dendritic cell maturation and their acquisition of the capacity to activate naive CD4 T cells.
Dendritic cells from TLR4-defective mice were similarly effective at
priming antigenically naive CD4 T cells for IFN- production in vitro
compared with dendritic cells from WT mice. In contrast,
allergen-specific IFN- production by CD4 T cells ex vivo was
moderately but significantly reduced for TLR4-defective mice compared
with WT. These different results, as far as IFN- production, may
reflect differences in the conditions for priming for allergen-specific
IFN- production by CD4 T cells in vitro vs in vivo. The in vitro
studies used a mixture of dendritic cells from lymph nodes, spleen, and
lung for in vitro priming of naive CD4 T cells over a 12-day period. In
vitro conditions may overcome certain limitations in priming for
cytokine production that would apply to the in vivo situation, such as
reduced production of IL-12 by dendritic cells or other APCs in the
lungs and draining lymph nodes of C3H/HeJ mice. Further studies of the
dendritic cell function of C3H/HeJ mice may help explain these
differences. Regardless, these results suggest that Th1 differentiation
from naive precursors is unaffected or only slightly reduced by a lack
of TLR4 signaling. They are also consistent with studies reporting that
C3H/HeJ mice are highly resistant to infection with certain
intracellular pathogens, such as Leishmania major, which
require CD4 Th1 cells for their effective control
(43).
The findings of this study are consistent with work by others that found that administration of LPS, a well-characterized TLR4 activator, resulted in enhanced Ag-specific IgE production and airway eosinophilia (19, 44) and that these effects were mediated by up-regulation of CD86 on APCs (44). This suggests that a TLR4 ligand, such as LPS derived from endogenous bacterial flora, may influence dendritic cell maturation in vivo, compromising the ability of these cells to up-regulation of costimulatory molecule expression and to direct Th2 immune responses. In support of a role for LPS derived from endogenous flora in increasing the capacity of dendritic cells to direct Th2 immune responses in vivo, we have also observed a similar phenotype of reduced experimental allergen-induced asthma and decreased Th2 responses in outbred mice raised under gnotobiotic conditions (K. Dabbagh and D. B. Lewis, unpublished observations). However, our results do not exclude other possibilities, including that defective TLR4 signaling impacts on Th2 responses by CD86-independent mechanisms involving dendritic cells. This is plausible given that other cell types that are important in Th2 pathology express TLR4, including mast cells (45) and CD4 T cells (46), and that LPS can enhance the production by mast cells of the Th2 cytokines IL-9 and IL-13 (47), both of which appear to be important in the immunopathogenesis of asthma (48, 49).
TLR4-defective dendritic cells also produced less of the IL-12 p40
subunit than WT cells in response to CD40 engagement, indicating an
additional immaturity in cell function in TLR4-defective mice and one
that is distinct from the expected impaired dendritic cell production
of IL-12 in response to LPS stimulation (50). This reduced
capacity for IL-12 production by dendritic cells was associated with
only a slight decrease in the ability of TLR4-defective mice to
generate allergen-specific IFN--producing CD4 T cells, indicating it
was not essential for the generation of Th1-like cells, at least in
this context. The biological importance of decreased production of
IL-12 by dendritic cells from TLR4-defective mice in allergic and
nonallergic contexts remains to be determined. Although endogenously
produced IL-12 has been reported to act as an attenuator of the
allergen-induced asthma in mice, based on studies using Ab
neutralization (21), enhanced Th2 responses were not
observed following allergen immunization in mice lacking IL-12 as a
result of selective gene targeting (51). Given that
endogenously produced IL-12 may actually contribute to the allergic
pulmonary response in mice, including eosinophilic inflammation
(52), it is plausible that reduced IL-12 production by
dendritic cells from TLR4-defective mice could actually contribute to
the reduced Th2 responses that we observed.
A direct interpretation of the results of this study is that activating signals through TLR4, most likely by LPS generated from commensal bacterial flora, are necessary for the maturation of the innate immune system so that it can optimally promote Th2-type immune responses to neoantigen. This interpretation is in agreement with some murine studies in which LPS administration increased allergen-specific IgE production and pulmonary eosinophilia (19, 44), but not another, in which genetic deficiency of LPS-binding protein, a molecule that serves as a chaperone for LPS binding to CD14, had Th2 responses similar to those of WT mice (53). Human epidemiological studies examining the role of LPS exposure in the risk of development of asthma have also been conflicting (22, 23). These conflicting results may be a reflection of the importance of the time and duration of exposure to LPS, as well as the immunological maturity of the subject in influencing the outcome for the immune response generated to an Ag (54, 55). Moreover, particular immunoregulatory effects of LPS on allergic disease could be dose dependent, a feature that is not modeled in the current study, in which mice with a complete genetic ablation of a major LPS-activating pathway were employed.
The finding that gene polymorphisms in the promoter of the CD14 gene are associated with increased serum soluble CD14 levels and with low serum levels of IgE (24) are also in contrast to our results obtained with TLR4-defective mice, if it is assumed that increased circulating CD14 results in enhanced LPS signaling. However, soluble CD14 may also serve as a shuttling molecule for transfer of LPS to circulating lipoproteins (56), a function that might reduce rather than enhance LPS-mediated effects via TLR4. Furthermore, CD14 is also involved in binding of ligands for TLR2 (1), so that the influence of CD14 polymorphisms on allergic disease in humans may not necessarily be mediated by effects on TLR4 activation. Interestingly, polymorphisms have also recently been identified in the human TLR4 gene, including some that result in amino acid alterations that decrease LPS responsiveness (57). Given the results of the current study, such TLR4 polymorphisms are strong candidates for influencing allergic diseases, such as asthma.
Finally, an alternative and nonexclusive possibility to account for our findings is that reduced dendritic cell maturation and allergen-induced pulmonary disease in C3H/HeJ mice reflects a lack of TLR4 activation by substances other than LPS, such as endogenously produced ligands. These potential ligands include heat shock protein 60, a stress-induced mitochrondrial matrix protein that activates mononuclear phagocytes (58) and dendritic cells (Ref. 59 and K. Dabbagh, unpublished observation) in a TLR4-dependent manner and enhances Ag-specific activation of naive CD4 T cells to cognate peptide in vitro (60). Fibrinogen and fibronectin have also recently been reported as activators of TLR4 (61, 62) and could contribute to increased inflammation in allergic pulmonary disease via a TLR4-dependent mechanism. Interestingly, respiratory syncytial virus F protein derived from respiratory syncytial virus has been reported to trigger the expression of proinflammatory cytokines by mononuclear phagocytes in a TLR4-dependent manner (63). This finding in conjunction with those of the current study raise the possibility of a previously unappreciated mechanism by which certain viral respiratory infections might act as cofactors for allergen-induced asthma. Therefore, additional work to define the role of various TLR4 activators in the development of allergen-induced asthma and the therapeutic potential of TLR4 blockade in this context, including following viral infection, will be of interest.
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Acknowledgments |
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Footnotes |
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2 Current address: Roche Bioscience, 3401 Hillview Avenue, M/S S3-1, Palo Alto, CA 94304.
3 Address correspondence and reprint requests to Dr. David B. Lewis, Center for Clinical Sciences Research Building, Room 2115b, 269 Campus Drive, Stanford University School of Medicine, Stanford, CA 94304-5164. E-mail address: dblewis{at}leland.stanford.edu
4 Abbreviations used in this paper: TLR, Toll-like receptor; WT, wild type; PCC, pigeon cytochrome c; i.n., intranasal; BAL, bronchoalveolar lavage.
Received for publication November 30, 2001. Accepted for publication March 12, 2002.
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References |
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B activation: a phylogenetically conserved paradigm in innate immunity. J. Clin. Invest. 107:13.[Medline]
T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction. Circulation 99:3218.2-microglobulin-dependent T cells are dispensable for allergen-induced T helper 2 responses. J. Exp. Med. 184:1507. and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944. production in T cells mediating chronic resistance to the intracellular pathogen, Toxoplasma gondii. J. Immunol. 165:628.B is decisively involved in the expression of IL-9. J. Immunol. 166:4391. production in T cells. Eur. J. Immunol. 31:2051.[Medline]
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K. Okunishi, M. Dohi, K. Fujio, K. Nakagome, Y. Tabata, T. Okasora, M. Seki, M. Shibuya, M. Imamura, H. Harada, et al. Hepatocyte Growth Factor Significantly Suppresses Collagen-Induced Arthritis in Mice J. Immunol., October 15, 2007; 179(8): 5504 - 5513. [Abstract] [Full Text] [PDF]
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 N. W. J. Schroder and M. Arditi IEIIS Meeting minireview: The role of innate immunity in the pathogenesis of asthma: evidence for the involvement of Toll-like receptor signaling Innate Immunity, October 1, 2007; 13(5): 305 - 312. [Abstract] [PDF]
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 Q. Wu, R. J. Martin, J. G. Rino, S. Jeyaseelan, R. Breed, and H. W. Chu A deficient TLR2 signaling promotes airway mucin production in Mycoplasma pneumoniae-infected allergic mice Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1064 - L1072. [Abstract] [Full Text] [PDF]
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 P. Winkler, D. Ghadimi, J. Schrezenmeir, and J.-P. Kraehenbuhl Molecular and Cellular Basis of Microflora-Host Interactions J. Nutr., March 1, 2007; 137(3): 756S - 772S. [Abstract] [Full Text] [PDF]
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 H. K. Parmentier, L. Star, S. C. Sodoyer, M. G. B. Nieuwland, G. De Vries Reilingh, A. Lammers, and B. Kemp Age- and Breed-Dependent Adapted Immune Responsiveness of Poultry to Intratracheal-Administered, Pathogen-Associated Molecular Patterns Poult. Sci., December 1, 2006; 85(12): 2156 - 2168. [Abstract] [Full Text] [PDF]
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A. M. Szema, S. A. Hamidi, S. Lyubsky, K. G. Dickman, S. Mathew, T. Abdel-Razek, J. J. Chen, J. A. Waschek, and S. I. Said Mice lacking the VIP gene show airway hyperresponsiveness and airway inflammation, partially reversible by VIP Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L880 - L886. [Abstract] [Full Text] [PDF]
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J. W. Hollingsworth, G. S. Whitehead, K. L. Lin, H. Nakano, M. D. Gunn, D. A. Schwartz, and D. N. Cook TLR4 Signaling Attenuates Ongoing Allergic Inflammation J. Immunol., May 15, 2006; 176(10): 5856 - 5862. [Abstract] [Full Text] [PDF]
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 Y. W. Jung, T. R. Schoeb, C. T. Weaver, and D. D. Chaplin Antigen and Lipopolysaccharide Play Synergistic Roles in the Effector Phase of Airway Inflammation in Mice Am. J. Pathol., May 1, 2006; 168(5): 1425 - 1434. [Abstract] [Full Text] [PDF]
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 L. A. Kerepesi, O. Leon, S. Lustigman, and D. Abraham Protective Immunity to the Larval Stages of Onchocerca volvulus Is Dependent on Toll-Like Receptor 4 Infect. Immun., December 1, 2005; 73(12): 8291 - 8297. [Abstract] [Full Text] [PDF]
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K. Okunishi, M. Dohi, K. Nakagome, R. Tanaka, S. Mizuno, K. Matsumoto, J.-i. Miyazaki, T. Nakamura, and K. Yamamoto A Novel Role of Hepatocyte Growth Factor as an Immune Regulator through Suppressing Dendritic Cell Function J. Immunol., October 1, 2005; 175(7): 4745 - 4753. [Abstract] [Full Text] [PDF]
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K. Nakagome, M. Dohi, K. Okunishi, Y. Komagata, K. Nagatani, R. Tanaka, J.-i. Miyazaki, and K. Yamamoto In Vivo IL-10 Gene Delivery Suppresses Airway Eosinophilia and Hyperreactivity by Down-Regulating APC Functions and Migration without Impairing the Antigen-Specific Systemic Immune Response in a Mouse Model of Allergic Airway Inflammation J. Immunol., June 1, 2005; 174(11): 6955 - 6966. [Abstract] [Full Text] [PDF]
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 R Lamb, E Zeggini, W Thomson, BSPAR, and R Donn Toll-like receptor 4 gene polymorphisms and susceptibility to juvenile idiopathic arthritis Ann Rheum Dis, May 1, 2005; 64(5): 767 - 769. [Abstract] [Full Text] [PDF]
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K. Okunishi, M. Dohi, K. Nakagome, R. Tanaka, and K. Yamamoto A Novel Role of Cysteinyl Leukotrienes to Promote Dendritic Cell Activation in the Antigen-Induced Immune Responses in the Lung J. Immunol., November 15, 2004; 173(10): 6393 - 6402. [Abstract] [Full Text] [PDF]
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D. V. R. Prasad, T. Nguyen, Z. Li, Y. Yang, J. Duong, Y. Wang, and C. Dong Murine B7-H3 Is a Negative Regulator of T Cells J. Immunol., August 15, 2004; 173(4): 2500 - 2506. [Abstract] [Full Text] [PDF]
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 H.-J. Anders, B. Banas, and D. Schlondorff Signaling Danger: Toll-Like Receptors and their Potential Roles in Kidney Disease J. Am. Soc. Nephrol., April 1, 2004; 15(4): 854 - 867. [Abstract] [Full Text] [PDF]
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S. C. Eisenbarth, A. Zhadkevich, P. Ranney, C. A. Herrick, and K. Bottomly IL-4-Dependent Th2 Collateral Priming to Inhaled Antigens Independent of Toll-Like Receptor 4 and Myeloid Differentiation Factor 88 J. Immunol., April 1, 2004; 172(7): 4527 - 4534. [Abstract] [Full Text] [PDF]
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 M. Kabesch and R. P. Lauener Why Old McDonald had a farm but no allergies: genes, environments, and the hygiene hypothesis J. Leukoc. Biol., March 1, 2004; 75(3): 383 - 387. [Full Text] [PDF]
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T. Darville, J. M. O'Neill, C. W. Andrews Jr., U. M. Nagarajan, L. Stahl, and D. M. Ojcius Toll-Like Receptor-2, but Not Toll-Like Receptor-4, Is Essential for Development of Oviduct Pathology in Chlamydial Genital Tract Infection J. Immunol., December 1, 2003; 171(11): 6187 - 6197. [Abstract] [Full Text] [PDF]
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S. C. Higgins, E. C. Lavelle, C. McCann, B. Keogh, E. McNeela, P. Byrne, B. O'Gorman, A. Jarnicki, P. McGuirk, and K. H. G. Mills Toll-Like Receptor 4-Mediated Innate IL-10 Activates Antigen-Specific Regulatory T Cells and Confers Resistance to Bordetella pertussis by Inhibiting Inflammatory Pathology J. Immunol., September 15, 2003; 171(6): 3119 - 3127. [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. S. Van Amersfoort, T. J. C. Van Berkel, and J. Kuiper Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clin. Microbiol. Rev., July 1, 2003; 16(3): 379 - 414. [Abstract] [Full Text] [PDF]
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 G. Lugo-Villarino, R. Maldonado-Lopez, R. Possemato, C. Penaranda, and L. H. Glimcher T-bet is required for optimal production of IFN-{gamma} and antigen-specific T cell activation by dendritic cells PNAS, June 24, 2003; 100(13): 7749 - 7754. [Abstract] [Full Text] [PDF]
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 S. C. Eisenbarth, D. A. Piggott, J. W. Huleatt, I. Visintin, C. A. Herrick, and K. Bottomly Lipopolysaccharide-enhanced, Toll-like Receptor 4-dependent T Helper Cell Type 2 Responses to Inhaled Antigen J. Exp. Med., December 16, 2002; 196(12): 1645 - 1651. [Abstract] [Full Text] [PDF]
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