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Departments of
* Microbiology and Immunology and
Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B of AMs and, unlike
OVA or LPS stimulation, up-regulated IL-6, TNF-
, and NO. In
addition, it down-regulated antioxidants, but affected neither the
expression nor production of IL-12. Der f-stimulated AMs expressed
enhanced levels of costimulatory B7 molecules, supported T cell
proliferation, and promoted Th2 cell development. The enhanced
accessory function was suppressed by blockade mAbs to B7.2, IL-6, and
TNF- and by N-monomethyl-L-arginine, an
NO synthase inhibitor, and N-acetylcysteine, a thiol
antioxidant, whereas it was augmented by
(±)-S-nitroso-N-acetylpenicillamine, an NO
donor. Arg-Gly-Asp-Ser peptide and neo-glycoproteins galactose-BSA and
mannose-BSA inhibited the Der f-induced IL-6 and TNF- productions
and enhanced accessory function of AMs. Der f was more potent than OVA
for inducing pulmonary eosinophilic inflammation, NO, and serum
allergen-specific IgG1 Ab production in mice. AMs from Der f-challenged
mice expressed enhanced levels of B7 and augmented T cell proliferation
ex vivo. In Der f-challenged mice, respiratory syncytial virus
infection (5 x 105 pfu; 3 days before Der f
instillation) augmented Der f-specific Ab production, whereas
dexamethasone (50 mg/kg; 1 h before Der f instillation) diminished
the allergic airway inflammation and Ab response. We conclude that AMs
are sensitive targets for Der f and that the Der f-induced
proinflammatory responses may represent an important mechanism in
mediating the development of allergic sensitization and
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alveolar macrophages
(AMs)3 are abundant in
the alveolar spaces, and they play a pivotal role in elimination of
foreign substances from the alveolar surface and initiation of lung
inflammation (3). Under normal steady-state conditions,
AMs function poorly as accessory cells, and their general actions in
the lung are suppressive for both T cells and pulmonary dendritic cells
(4). The inhibitory activity of AMs occurs at an early
stage in T cell activation (5), which might be related to
a variety of secreted mediators including leukotrienes, prostaglandins,
immunosuppressive cytokines (such as IL-10 and TGF-
), and NO
(6, 7). In addition, AMs are important in prevention of
allergic sensitization in the healthy lung. Depletion of AMs can lead
to a vigorous immune response with influx of T cells and IgE Ab
production in allergic mice (8). In asthmatic subjects,
the allergic airway inflammation was associated with functional changes
of AMs, including an altered phenotype pattern (9), an
enhanced Ag presentation capacity (10), and an increased
production of secretory products (including superoxide anion and
cytokines) (11, 12). Furthermore, AMs from atopic
asthmatic subjects differentially regulated allergen-specific Th2- but
not Th1-type responses (13) and enhanced IL-5 production
by CD4+ T cells after stimulation with allergen
or mitogen (14). These previous studies clearly
demonstrated that AMs participate in the pathogenesis of asthma.
However, the role of AMs in allergic sensitization remains to be
established.
House dust mite (HDM) is a major source of environmental inhaled allergens. In vitro studies have shown that HDM allergens affect a variety of cell types including bronchial epithelial cells (15), mast cells (16), T cells (17), and B cells (18). In addition, it can activate both the kallikrein (19) and complement systems (20). These properties of HDM allergens are thought to be associated with their allergenicity. Although not well characterized, it has been shown that the whole mite extract of Dermatophagoides pteronyssinus binds to human lung surfactant protein A and D, which are members of the collectin family of the C-type lectins (21). The binding was abrogated after deglycosylation, suggesting that carbohydrate moiety may be involved in the interaction. However, there is little information relating the biological sequelae of inhalation of HDM allergens that result in allergic sensitization. Previously, we demonstrated that a crude extract of D. farinae (Der f) could induce acute pulmonary inflammation with increased proinflammatory cytokine levels in mice after a single intratracheal (i.t.) inoculation. The animals then developed a Th2 polarized allergic response with the characteristics of bronchial asthma after repeated challenge (22). These results suggest that Der f may possess certain properties that are prone to induce allergic airway inflammation and sensitization in the host. Because AMs express a large number of surface receptors, including those that have specificities for sugar residues commonly found in nonmammals such as bacteria and fungi, we assume that AMs may be involved in early recognition of HDM allergens after inhalation into the lung. In addition, the biological activities of the allergens may be involved in the development of the subsequent allergic responses. In the present study, we examine whether AMs represent a target of Der f action and to what extent the proinflammatory activity of Der f contributes to the development of allergic response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free, female, 6- to 8-wk-old BALB/c mice (Laboratory Animal Center, National Cheng Kung University, Tainan, Taiwan, Republic of China) were used unless otherwise indicated. Animals were fed sterile food and water ad libitum. All experimental animal care and treatment followed the guidelines set up by the Institutional Animal Care and Use Committee.
Reagents
Der f (1 g lyophilized whole body extract in ether; Allergon, Engelholm, Sweden) (22), OVA, or LPS (Escherichia coli 055:B5; both from Sigma-Aldrich, St. Louis, MO) were dissolved in pyogenic-free isotonic saline, filtered through a 0.22-µm filter, and stored at -70°C before use. LPS concentration of the Der f preparations was <0.96 EU/mg of Der f (Limulus amebocyte lysate test; E-Toxate; Sigma-Aldrich). Arg-Gly-Glu-Ser (RGDS) peptide (American Peptide Company, Sunnyvale, CA), galactosylated and mannosylated BSA (EY Laboratories, San Mateo, CA), N-monomethyl-L-arginine (L-NMMA), (±)-S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem-Novabiochem, La Jolla, CA), N-acetylcyteine (NAC), dexamethasone, and polymyxin B (Sigma-Aldrich) were each dissolved in PBS before use. Respiratory syncytial virus (RSV; A2 strain) was obtained from the National Health Research Institute Virology Laboratory for Diagnosis and Research at the National Cheng Kung University Hospital. The stock was grown and assayed for infectivity in HEp-2 cells. These cell cultures were maintained in DMEM supplemented with 10% (v/v) FBS, penicillin (100 IU/ml), and streptomycin (100 µg/ml). Stock contained 106 pfu/ml of infective virus.
Allergen sensitization and assessment of blood eosinophilia and airway inflammation
Groups of six mice were i.t. inoculated with five doses of Der f or OVA (0.5 mg/ml, 50 µl) at 1-wk intervals according to the method previously described (22). At 1, 3, or 7 days after the last challenge, the numbers of blood eosinophils were determined using a diagnostic reagent system (Unopette test 5877; BD Biosciences, Rutherford, NJ) with blood samples collected via the orbital sinus under light anesthesia. Mice were then killed by i.p. injection of overdose sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL), and serum samples were collected and stored at -70°C until assay. Bronchoalveolar lavage (BAL) was performed (two washes of 1 ml of ice-cold endotoxin-free saline each) as described previously (22). The BAL fluids were separated (2000 rpm, 10 min) and stored at -70°C. After total leukocyte counting, differential counts were performed on cytospin preparations (2 x 104 cells/100 µl BAL fluid) stained with Liu stain (Tonyar Diagnostics, Taipei Hsien, Taiwan) in a blind manner. BAL cells were then pooled for further assays. For inhibition studies, mice were i.p. injected with dexamethasone (50 mg/kg) 1 h before each Der f inoculation. For RSV infection, mice were i.t. infected with RSV (5 x 105 pfu) 3 days before each inoculation and were examined at day 3 after the last challenge.
Flow cytometric analysis
Pooled BAL cells (1 x 106) were incubated with anti-CD16/CD32 mAb (Fc blocker, 2.4G2) followed by FITC-conjugated anti-B7.1 (16-10A1) or anti-B7.2 (GL1) and PE-conjugated anti-I-Ad (AMS-32.1) mAbs (BD PharMingen, San Diego, CA) for 30 min on ice. After washing, stained cells were quantified by using FACScan (BD Immunocytometry Systems, San Jose, CA). Isotype-matched mAb-stained cells were used as a background control in all experiments.
Allergen-specific IgG1 and IgG2a/2b
Serum samples (1:5 dilution) were added in duplicate onto ELISA plates coated with Der f or OVA (20 µg/ml in 0.1 M NaHCO3, pH 8.3). After incubation overnight at 4°C, the plates were washed and incubated with biotinylated rat anti-mouse IgG1 (A85-1) or IgG2a/2b (R2-40) mAb (2 µg/ml; BD PharMingen) for 1 h, followed by washings and the addition of streptavidin-HRP conjugate (1:1000; BD PharMingen). The plates were washed and developed with TMB microwell peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Absorbance of the samples was determined at 450 nm.
Stimulation of AMs
BAL cells from normal mice (2 x 105) were seeded on 96-well culture plates and incubated for 1 h at 37°C and 5% CO2. Residual nonadherent cells (<5%) were removed, and adherent BAL macrophages (>95% viability) were cultured in triplicates in RPMI 1640 supplemented with 10% FBS, 20 mM L-glutamine, 10 mM sodium pyruvate, 1% nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. Cells were then treated with medium alone or with 10 µl of predetermined noncytotoxic doses of Der f, OVA, or LPS. Supernatants were collected after 24-h culture. After extensive washing, cells were coincubated with T cells or subjected to other analyses. For heat treatment, Der f was heated for 5 min at 100°C before use. For inhibition studies, AMs were pretreated with various inhibitors for 40 min, washed, and then incubated with Der f.
Coculture of AMs and T cells
AMs were recovered by BAL from allergen-challenged mice or from
normal mice and then stimulated with the allergens as described above.
Autologous plastic-nonadherent splenic cells (primarily lymphocytes)
were collected and purified by nylon wool column separation.
Single-cell suspensions of splenic T cells (2 x
105) were then cocultured with the AMs (2 x
104) in the presence of PHA (1 mg/ml; Murex
Biotech Limited, Dartford, U.K.). After 24 h of incubation, 0.5
µCi of [3H]thymidine (Amersham Life Science,
Buckinghamshire, U.K.) was added and further incubated for 24 h.
Cells were then harvested, and radioactivity as cpm was determined by
liquid scintillation counting. In inhibition experiments, AMs were
stimulated with Der f in the presence of the following reagents: mAb to
B7.1 (16-10A1), B7.2 (GL1), IL-6 (MP5-20F3), TNF- (G281-2626) (5
µg each), L-NMMA, SNAP (200 µg each), NAC (25 µM), or
dexamethasone (100 µg). Counts per min were determined 24 h
after the addition of T cells. For T cell phenotyping, the cocultures
were incubated for 24 h without the addition of isotopes. Then
supernatants were collected and levels of IL-4 and IFN-
were
determined by immunoassay as described below.
ELISA of cytokine levels
Concentrations of cytokines were measured by a sandwich
ELISA technique using commercial matching mAb pairs, one of which was
biotinylated (IL-6, MP5-20F3 and MP5-32C11; TNF-, G281-2626 and
MP6-XT3; IL-12, C15.6 and C17.8; IL-4, 11B11 and BVD6-24G2;
IFN-, R4-6A2 and XMG1.2; BD PharMingen). The reaction was revealed
and developed as described above. Standards were run in parallel with
recombinant cytokines. The detection limits were 7.5 pg/ml for IL-6,
IL-4, and TNF- and 15 pg/ml for IL-12 and IFN-.
Semiquantitative RT-PCR
RT-PCR was performed to estimate mRNA expression of cytokines
and inducible NO synthase (iNOS). Total cellular RNA was extracted from
pooled AM samples (RNeasy Total RNA kit; Qiagen, Hilden, Germany) and
converted to cDNA with StrataScript H-reverse transcriptase
(Stratagene, La Jolla, CA). PCR amplification (GeneAmp PCR System 2400;
PerkinElmer, Branchburg, NJ) was performed on a 1-µl cDNA sample.
Amplification cycle numbers and annealing temperatures were optimized
for each primer pair. PCR products were electrophoresed on 2% agarose
and stained with ethidium bromide. To control for sample-to-sample
variation, the amount of cDNA input to all samples was first adjusted
to comparable levels of -actin transcripts before it was subjected
to PCR amplification. Gene-specific primer pairs (sense and antisense,
respectively) were used as follows: TNF-,
5'-AGCCCACGTCGTAGCAAACCACCAA-3' and
5'-ACACCCATTCCCTTCACAGAGCAAT-3'; IL-1, 5'-TCATGGGATGATGATAACCTGCT-3'
and 5'-CCCATACTTTAGGAAGACACGGATT-3'; IL-6,
5'-CTGGTGACAACCACGGCCTTCCCTA-3' and 5'-ATGCTTAGGCATAACGCACTAGGTA-3';
IL-12, 5'-GCAAGAGACACAGTCCTGGG-3' and 5'-TGCATCAGCTCATCGATGGC-3';
RANTES, 5'-GTACATCACCATGGCGTATG-3' and
5'-TCTTCTCTGGGTTGGCACACA-3'; TGF-1,
5'-GCGGACTACTATGCTAAAGATG-3' and 5'-GTTGTGTTGGTTGTAGAGGGC A-3';
iNOS, 5'-AAGTCAAATCCTACCAAAGTGA-3' and
5'-CCATAATACTGGTTGATGAACT-3'; and -actin,
5'-TGGAATCCTGTGGCATCCATGAAAC-3' and
5'-TAAAACGCAGCTCAGTAACAGTCCG-3'.
Nuclear extracts and EMSA
Nuclear extracts were prepared from AMs (1 x
106) exposed to Der f for 24 h by the method
of Feng and Lo (23). Consensus oligonucleotides of NF-B
(5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega, Madison, WI) were 5'
end-labeled with T4 polynucleotide kinase (Promega) and
[-32P]ATP (Amersham Life Science). Binding
reaction consisted of 10 µg of nuclear extracts and 0.5 ng of DNA
probe. Supershift experiments were performed using polyclonal Abs to
the p65 or p50 of NF-B (Santa Cruz Biotechnology, Santa Cruz, CA).
Competitive assays with unlabeled oligonucleotides used a 100-fold
molar excess relative to the radiolabeled oligonucleotides.
Analysis of NO
NO contents were determined using capillary electrophoresis as described by Bories et al. (24), with modification. This method allows simultaneous determination of nitrite and nitrate in biological fluids and is used as a marker of NO production. Electrophoresis was conducted in a 67-cm long, 50-µm diameter silica-fused capillary at 12 kV with on-column UV detection at 214 nm. The separation buffer was 20 mM sodium sulfate containing a positively charged surfactant, tetradecyltrimethyl-ammonium bromide (Sigma-Aldrich). Under these conditions, the two anions migrated in the order of nitrite and nitrate (5.2 and 5.5 min, respectively). The lower detection limit was 1 µM, and the linearity was up to 400 µM.
Measurement of antioxidant activity
Total antioxidant activity was determined on an AutoLumat LB 953
Luminometer (EG & Berthold, Wildbad, Germany) according to the method
described by Whitehead et al. (25). In brief, samples (10
µl) were introduced to a chemiluminescent reaction generated by
mixing signal reagent (luminol and p-iodophenol, 100 µl)
and HRP solution (50 µl, 1:200 dilution) in distilled water (800
µl). The duration of the quenching was measured and compared with
that of a water-soluble -tocopherol analog, trolox
(6-hydroxy-2,5,7,8-tetra-methychroman-2-carboxylic acid). Precision of
the assay was 2.3% and 5.1% for within-day and day-to-day variation,
respectively.
Statistical analyses
Mediator contents in culture supernatants and T cell proliferation values were analyzed by one-way ANOVA test, followed by a post hoc comparison between groups. The cellular accumulation and mediator contents in BAL fluid and serum Ab titer were analyzed using unpaired t test. Results are expressed as means ± SEM. A p value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first tested whether Der f could stimulate AMs to release
proinflammatory mediators. Freshly isolated AMs were stimulated with
Der f for 24 h. OVA, a stereotypical allergen, and LPS were used
as controls. AMs stimulated by Der f produced large amounts of IL-6 and
significant concentrations of TNF- and NO (combined nitrite and
nitrate levels), with an undetectable level of IL-12 (Fig. 1
A). However, Der f
significantly suppressed antioxidant production. Semiquantitative
RT-PCR showed that the Der f dose dependently increased the expression
of the mRNA for TNF-, IL-1, IL-6, RANTES, and iNOS in AMs (Fig. 1B), whereas it did not induce IL-12 and it markedly
repressed TGF-1 expression. EMSA revealed NF-B activation in AMs
after Der f stimulation (Fig. 1C). Under the same
experimental settings, OVA augmented IL-6 and TNF- productions at
high dosage (10 µg), affected neither NO nor antioxidant production,
and only moderately induced the mRNA expression of AMs. In contrast,
LPS enhanced the expressions of all the cytokines and iNOS and
augmented IL-12 as well as IL-6, TNF-, and NO productions. These
results indicate that Der f mobilizes a variety of proinflammatory
mediators of AMs and that the mediator profile was dissimilar to those
induced by OVA or LPS.
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Naive AMs have a suppressive nature for T cell activation. Thus,
we next examined the accessory function of Der f-stimulated AMs on
mitogen-induced T cell proliferation. As expected, naive autologous AMs
markedly inhibited T cell proliferation in response to PHA (25–30% of
control T cells; Fig. 2A). The
AMs’ suppressive activity was lost in a dose-dependent manner after
prestimulation with Der f for 24 h. In other words, Der
f-stimulated AMs promoted T cell proliferation. AMs obtained from
C57BL/6 and LPS-resistant C3H/HeJ mice exhibited a similar response,
whereas OVA-stimulated AMs and Der f itself had no effect. Flow
cytometric analysis showed a 2-fold increase in B7.1 and a 2- to 4-fold
increase in B7.2 expression on Der f-stimulated AMs, compared with a
small increase in B7.1 and no increase in B7.2 expression on
OVA-stimulated AMs (Fig. 2B). To further clarify the effect
of Der f on the Th1/Th2 balance, we measured IL-4 and IFN- in the
supernatants of the cultures. Compared with samples with nonstimulated
AMs, significantly higher levels of IL-4 were detected in cultures with
Der f-stimulated AMs (Fig. 3). On the
contrary, cultures containing OVA- or LPS-stimulated AMs had increased
levels of IFN-, but not IL-4. These results indicate that Der f
augments the accessory function of AMs and that Der f-stimulated AMs
facilitate the development of Th2 cells.
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Blocking mAbs to B7 molecules and cytokines, a NOS inhibitor, and
an antioxidant were subsequently used to study the requirements for T
cell proliferation. It was found that mAb to B7.2, but not to B7.1 or
isotype control mAbs, significantly suppressed the T cell proliferation
induced by Der f-stimulated AMs (the mean ± SEM of percent
inhibition was 37 ± 3%; p < 0.05; Fig. 4A). In addition,
L-NMMA, a NOS inhibitor, and NAC, a thiol
antioxidant, also markedly inhibited the proliferation (percent
inhibition, 54 ± 3% and 82 ± 2%, respectively;
p < 0.05; Fig. 4B). In contrast, SNAP, an
NO donor, regained the repressed proliferation induced by
L-NMMA. Neutralizing mAbs to IL-6, TNF-, or a
combination of both, and dexamethasone all effectively reduced the
proliferation (percent inhibition, 61 ± 2%, 69 ± 1%,
72 ± 1%, and 81 ± 4%, respectively; p <
0.05; Fig. 4C). Thus, Der f-stimulated AMs expressed
functional B7.2 molecules, and secreted NO, oxidants, and
proinflammatory cytokines were important in the induction of T cell
proliferation.
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Based on the previous demonstration (21) that
surfactant protein A and D bind to the carbohydrates of D.
pteronyssinus, we next investigated the potential role of both
lectin and integrin receptors in Der f and AM interaction. AMs were
incubated with Der f after the pretreatment with increasing
concentrations of potential inhibitors. The peptide RGDS, an inhibitor
of 2 integrins, significantly inhibited the IL-6 and TNF-
productions of Der f-stimulated AMs in a dose-dependent manner (percent
inhibition at 50 µg/ml: 79 ± 3% and 62 ± 3%,
respectively; p < 0.05; Table I). Incubation with galactose-BSA or
mannose-BSA also inhibited TNF- production (percent inhibition at 50
µg/ml: 59 ± 2% and 62 ± 7%, respectively;
p < 0.05). Moderate inhibition of IL-6 was only seen
at high dosage of galactose-BSA (percent inhibition: 32 ± 5%;
p < 0.05). In addition, RGDS, galactose-BSA, and
mannose-BSA (50 µg each) suppressed T cell proliferation induced by
Der f-stimulated AM in a range of 50–60% (Table II). The inhibitors themselves did not
affect the proliferation. Furthermore, neither heat treatment (100°C,
5 min) nor polymyxin B affected the Der f-induced cytokine production
of AMs (Table I). These results suggest that the carbohydrate moiety of
the mite extracts may bind to AM 2 integrins
and C-type lectins.
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To further explore the proinflammatory activity of Der f, we
investigated Der f challenge responses and sensitization in mice in
comparison with OVA. We used a sensitization regimen with repetitive
i.t. instillation, for which the effect of Der f challenge has
partially been characterized (22). Analysis of BAL fluids
revealed that repetitive challenge induced a cellular influx into the
airways, irrespective of the type of allergens (Fig. 5A). In Der f mice, the
numbers of total BAL cells peaked at day 1, lasted up to 7 days
post-challenge, and consisted of increased numbers of neutrophils,
lymphocytes, and eosinophils, which were significantly higher and
sustained longer than those of OVA mice. There was a 15- to 25-fold
increase in NO and a 50–75% decrease in antioxidant concentrations in
BAL fluids of Der f mice (Fig. 5B). In contrast, NO was not
increased in the BAL fluids of OVA mice, and the levels of antioxidant
were moderately decreased (35–40%) at days 1 and 3 post-challenge.
Blood eosinophil counts were significantly higher in Der f mice than in
OVA mice at day 7 after challenge. Induction of sensitization was
evaluated by allergen-specific Ab production. Der f mice produced high
levels of Der f-specific IgG1 Ab, which is normally associated with a
Th2 immune response. In contrast, OVA-specific IgG1 and IgG2a/2b Ab
titers were elevated in OVA mice, suggesting a mixed Th1/Th2 response
(Fig. 5B). These results indicate that Der f can manifest
its proinflammatory activity in vivo and that it may have certain
properties that are prone to induce a Th2 response.
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To further clarify the effect of Der f on AMs, the accessory
function of AMs from allergen-challenged mice was examined ex vivo. In
concert with the in vitro finding, AMs obtained from Der f mice lost
their suppressive activity and augmented T cell proliferation (Fig. 6A). The enhancement effect
was observed 1 day after the last challenge, was maximal at day 3, and
returned to baseline levels at day 7. This pattern was correlated with
the course of the airway inflammatory responses and an up-regulation of
B7.2 on AMs (Fig. 6B). On the contrary, AMs of OVA mice
retained their suppressive activity and showed no enhanced B7.2
expression.
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To investigate the contribution of the proinflammatory activity of
Der f, as well as an coexisting airway inflammation in Der f
sensitization, we either treated mice with an i.p. injection of
dexamathasone at Der f challenge or infected them with live RSV 3 days
before the challenge. Our experience and Matsuse et al.
(26) have demonstrated that RSV infection elicits an
airway inflammation in mice. Dexamathasone, an NF-B inhibitor,
effectively blocked the Der f-induced responses, including blood and
pulmonary eosinophilia (Fig. 7),
up-regulation of NO, and repression of antioxidant in BAL fluids (data
not shown). Furthermore, Der f-specific Ab productions were almost
completely prevented by this treatment. A preinfection of RSV did not
deteriorate the Der f-induced airway inflammation. Total and
differential counts of BAL cells were compatible between RSV-infected
and noninfected challenged mice, except that there were higher
lymphocyte counts in the infected mice (Fig. 7). RSV infection,
however, affected the systemic effect of Der f. The numbers of blood
eosinophils and Der f-specific Ab titers were higher in RSV-infected
challenged mice than in noninfected challenged mice. These results
indicate that an airway inflammatory response, either provoked by Der f
itself or by a concomitant RSV infection, is essential for Der f
sensitization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, mobilized a variety of proinflammatory mediators, and
up-regulated B7 expression of AMs after 24-h incubation. Der
f-stimulated AMs, in turn, became more effective at supporting T cell
proliferation and promoting Th2 cell development. Der f activities were
heat stable and could be inhibited by RGDS peptide and by galactose-BSA
and mannose-BSA, indicating the involvement of the carbohydrate moiety
of Der f and the integrin receptors and C-type lectin of AMs in
mediating the binding. Furthermore, AMs isolated from Der f-sensitized
mice exhibited a similar functional change ex vivo (up-regulation of B7
molecules and an enhanced accessory function), suggesting the
involvement of AMs in innate recognition of HDM allergens in the lung.
The Der f-induced functional changes of AMs may represent an early
cellular response in the sensitization process, which can then regulate
the character and magnitude of the allergic reactions.
Initially, while investigating the mediator-inducing capacity of Der f,
we found that both Der f and LPS had pronounced mediator-inducing
potential and were able to stimulate AMs to produce IL-6, TNF-, and
NO. However, unlike LPS, Der f induced neither the expression nor
production of IL-12, a key cytokine for Th1-type response
(27). This discrepancy can be explained by the fact that
the two stimuli use different receptors on AMs. LPS is known to
activate monocytes and macrophages via the Toll-like receptor 4,
leading to the production of cytokines (28). Der f may
activate AMs, although not exclusively, through integrin receptors and
C-type lectin. Similarly, a previous study showed that AMs bound and
phagocytosed allergenic pollen starch granules via C-type lectin and
integrin receptors, resulting in up-regulation of iNOS and release of
NO (29). In our study, OVA excreted a relatively weak
mediator-inducing capacity. In fact, many studies have shown that OVA
is unable to induce gross airway inflammation in murine model of asthma
(30, 31). In addition to the distinct mediator profile,
several lines of evidence indicate that the Der f effects were
unrelated to LPS. First, the Der f preparations contained very low
levels of LPS (<0.96 EU/mg Der f). Second, AMs derived from C3H/HeJ
mice responded to Der f as well. Third, polymyxin B did not affect the
Der f activity. House dust has been shown to contain large amounts of
endotoxin that are probably related to the severity of asthma
(32). Thus, it is significant to determine to what extent
LPS influences the AM responses to Der f, because that information will
advance our overall understanding of the allergenicity of HDM allergens
in house dust. Furthermore, we found that Der f could activate NF-B.
The role of NF-B in the induction of airway eosinophilia has been
demonstrated in mice lacking the p50 subunit (33).
A significant finding was that in Der f-stimulated AMs, the proinflammatory cytokines were coexpressed with NO, whereas antioxidants were down-regulated. Increased NO production by the airways is associated with asthma, and exhaled NO has been proposed as a clinically useful marker of asthmatic airway inflammation (34). Furthermore, loss of superoxide dismutase has been observed within minutes after segmental Ag instillation into the lungs of asthmatic subjects (35). However, the role of both NO and antioxidants in the pathogenesis of asthma still remains incompletely understood. NO is well known to regulate cytokine gene expression and to influence immune and inflammatory responses (36). Studies also have suggested that an autoregulatory feedback loop exists between NO and proinflammatory cytokines (37). Furthermore, the physiologic consequence of NO produced in the airways is critically dependent upon the chemical milieu of the surrounding environment, especially the balance between oxidants and antioxidants (38). In this context, the Der f-induced proinflammatory milieu, namely high levels of NO and IL-6, low level of antioxidants, and absence of IL-12, might represent an essential regulatory mechanism in the lung microenvironment during allergic sensitization.
In physiological conditions, AMs act as a suppressor of immune
responses in the lung (3, 4). However, AMs obtained from
atopic asthmatics have been shown to enhance rather than suppress PBMC
proliferation (14), and AMs differentially regulated
allergen-specific Th2- but not Th1-type responses (13),
indicating that AMs participate in the pathogenesis of asthma.
Significantly, we observed a similar functional change of AMs after Der
f stimulation. Der f- but not OVA- or LPS-stimulated AMs supported T
cell proliferation and promoted naive T cell acquisition of a Th2 cell
phenotype. We speculate that this homeostatic modulation of AMs may
allow unchecked proliferation of T cell and thus may accelerate the
sensitization process. These results also prompted us to clarify
whether the Der f-induced mediators played a role in mediating the
accessory function of AMs. We found that the enhanced accessory
function of Der f-stimulated AMs was dependent on B7.2 costimulation,
which is known to be essential for the development of Th2 response
(39). Thus, Der f up-regulated B7.2 of AMs, which in turn
promoted T cell response. Interestingly, blockade of the Der f-induced
mediators, including IL-6, TNF-, and NO, was even more effective in
inhibiting the accessory function. NO by itself is an inhibitor of T
cell proliferation, whereas NO’s inhibitory activity can be
neutralized by simultaneous superoxide production by macrophages
(40). Thus, we presume that Der f also stimulated reactive
oxygen species production of AMs. The effectiveness of NAC in
inhibiting the proliferation supported this assumption. Indeed, our
recent study has confirmed the production of reactive oxygen species by
AMs after Der f stimulation (C. K. Yu and C. T. Lee, unpublished
observation). The role of proinflammatory cytokines in mediating
the response, however, is not fully understood at the present time.
APC-derived IL-6 was able to initiate the polarization of naive
CD4+ T cells to effector Th2 cells
(41). Alternatively, the cytokines might affect the
accessory function indirectly by modulating the expression of
costimulatory molecules or other mediators (37). Again,
our results support our aforementioned assumption that the mediators
secreted by the Der f-stimulated AMs would modulate the accessory
function of AMs, perhaps in an autocrine or paracrine manner, and that
the overall activities of the Der f-stimulated AMs favor Th2 cell
development.
Studies have shown that innate immunity modulates the asthmatic response (42, 43). Furthermore, the development of allergic responses to OVA in murine model of asthma could be facilitated by an airway inflammation caused by viral infection (30) or a diesel exhaust particle-mediated oxidative stress (31). These indicate that airway inflammatory mechanisms play a vital role in the pathogenesis of asthma. Thus, it is important to determine to what extent the proinflammatory activities of Der f contribute to allergic sensitization. Using a sensitization regimen with repetitive challenge (22), we demonstrated that Der f, in concert with its in vitro mediator-inducing capacity, was more effective than OVA in eliciting allergic inflammation and sensitization in mice. In addition, a concurrent RSV infection, which can provoke an acute airway inflammation, could promote Der f-specific Ab production in sensitized mice. In agreement with previous reports (30, 31), our data fully support the notion that a coexisting airway inflammation is an asset for allergic responses. In studies aimed at inhibiting the proinflammatory activities of Der f, we found that administering dexamethasone at the time of Der f challenge markedly attenuated the allergic airway inflammation and resulted in failure of allergic sensitization. Although it may not be a direct proof, the data indeed highlight the essence of the proinflammatory activities of Der f on the sensitization process. Furthermore, we found that AMs obtained from mice that were challenged by Der f but not by OVA exhibited a response ex vivo similar to those stimulated by Der f in vitro, indicating that AMs not only responded to Der f in vitro but that they also functioned as effector cells in Der f-challenged mice. Nevertheless, we cannot exclude contributions made by other cell types (for example, mast cells, dendritic cells, and epithelial cells) (15, 16) or the complement and kinin systems (19, 20) in mediating the tissue responses.
Taken together, these results demonstrate an array of immunomodulation and proinflammatory activities of Der f on AMs, which may be considered as essential factors for their allergenicity. An enhanced AM accessory cell function with a generation of a Th2-prone proinflammatory milieu may represent an important mechanism for initiating and amplifying allergic inflammation and sensitization.
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Acknowledgments |
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2 Address correspondence and reprint requests to Dr. Chun-Keung Yu, Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 70101, Republic of China. E-mail address: dckyu{at}mail.ncku.edu.tw
3 Abbreviations used in this paper: AM, alveolar macrophage; HDM, house dust mite; Der f, Dermatophagoides farinae; i.t., intratracheal; RGDS, Arg-Gly-Glu-Ser; L-NMMA, N-monomethyl-L-arginine; SNAP, (±)-S-nitroso-N-acetylpenicillamine; NAC, N-acetylcyteine; RSV, respiratory syncytial virus; BAL, bronchoalveolar lavage; iNOS, inducible NO synthase.
Received for publication June 11, 2002. Accepted for publication October 25, 2002.
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and interleukin-6 by alveolar macrophages consecutive to the development of the late asthmatic reaction. J. Allergy Clin. Immunol. 88:561.[Medline]
subunit of the human T cell interleukin 2 receptor, by Der p 1, a major mite allergen with cysteine protease activity. J. Exp. Med. 187:271.B and activator protein 1. Infect. Immun. 67:2951. production and diminishes IL-4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.B in the induction of eosinophilia in allergic airway inflammation. J. Exp. Med. 188:1739.B activation in alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 21:311. and interleukin-4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. J. Clin. Invest. 100:829.[Medline]
T cells in allergic airway inflammation. Science 280:1265.This article has been cited by other articles:
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