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* Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037; and
Department of Pharmacology, University of Illinois, Chicago, IL 60612
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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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Two subtypes of kinin receptors, the B2 receptor and the B1 receptor, were originally distinguished based upon functional data in animal tissues (9). In humans, cloned gene sequences of the receptors have confirmed distinct B2 and B1 receptors, each belonging to the seven transmembrane G protein-coupled superfamily (10, 11). BK and Lys-BK (kallidin) are equally effective agonists for the B2 receptor; however, the carboxypeptidase metabolite of Lys-BK, Lys-des-Arg-BK, is the only known potent agonist for the human B1 receptor (11, 12). B2 receptors are constitutively expressed on many cell types (13) including human airway epithelial cells (14), whereas B1 receptors are expressed at low levels in normal tissues but can be induced in response to pathophysiological stimuli (15, 16, 17).
In the human airway, the majority of evidence has supported mediation of kinins’ effects occurring through the B2 receptor. Inhalation of BK or Lys-BK provoke acute bronchoconstriction (18). Icatibant (HOE-140), a B2 receptor antagonist, abolished hyperresponsiveness to histamine and reduced nasal eosinophilia induced by Ag in subjects with allergic rhinitis (19). In asthmatic subjects treated with icatibant, pulmonary function gradually improves over a 4-wk treatment phase, suggesting that blockade of the B2 receptor also had an anti-inflammatory role (20). In contrast, human airway provocation with Lys-des-Arg-BK failed to cause either bronchoconstriction in asthmatic subjects or increased glandular secretion and vascular permeability in the upper airways of subjects with allergic rhinitis (21). Participation of the B1 receptor in human airway disease is thus uncertain. However, in an allergen rat model a B1 receptor antagonist inhibited allergen-induced bronchial hyperresponsiveness (22).
In the current investigation, we sought to better define the potential role of B1 receptors in human airway inflammation. We examined the expression of B1 receptor mRNA in nasal tissue from subjects with allergic rhinitis compared with normal controls, and measured the effects of nasal allergen challenge on B1 receptor mRNA expression. Finally, we assessed the ability of Lys-des-Arg-BK to transduce in vivo activation of a mitogen-activated protein kinase (MAPK) in nasal tissue, and investigated the effect of this ligand on transcription factor activation in cultured airway epithelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonsmoking adults with allergic rhinitis (diagnosis based on a consistent history and confirmatory aeroallergen skin tests) and normal controls were recruited to participate. Informed consent was obtained in accordance with the Human Subjects Committee at The Scripps Research Institute (La Jolla, CA). Allergic rhinitis subjects undergoing nasal challenges were excluded if they had received prior immunotherapy, and all allergy and or asthma medications were held for at least 2 wk before challenge. Participants were screened with prick skin tests for common aeroallergens (Greer Laboratories, Lenoir, NC). A positive reaction was defined as a wheal diameter at least 3 mm greater than the saline control. Screening panels included grass mixture, Bermuda grass, tree mixture, weed mixture, dust mite (Dermatophagoides pteronyssinus and Dermatophagoides farinae), cat, and dog.
Nasal challenge protocols
Allergen challenge. Subjects with mild or quiescent allergic rhinitis underwent graded nasal allergen challenge to establish the provoking dose for symptomatic rhinitis. Six subjects were challenged with mite (D. farinae and D. pteronyssinus), two with cat, and seven with grass allergen. Subjects were asked to record their global severity score using a visual analog scale with 0 for none and 10 for severe. Subjects were questioned for the appearance of typical symptoms of allergic rhinitis including sneezing, itching, burning, congestion, rhinorrhea, throat irritation, and ocular symptoms of itching, tearing, or burning. Allergen was delivered by gently insufflating 100 µl aliquots into the nasal passage using a tuberculin syringe that was placed in the nostril. The dose of allergen was increased at 10-fold increments every 5 min from an initial 1/100,000 dilution of concentrated extract until symptoms were elicited of 5 or greater on the visual analog scale. The cumulative dose of allergen delivered into the nare eliciting the symptomatic response was then designated as the provoking dose. For pollen sensitive subjects, challenges were conducted out of season.
One month after the provoking dose was determined, subjects returned for a baseline nasal sample. Subsequent nasal samples were obtained using one of two protocols. Subjects studied using the first protocol had a baseline nasal sample obtained, then returned for allergen challenge 1 wk later. At that time, each subject underwent bilateral nasal allergen challenge using the provoking dose delivered in each nare. Two timed nasal samples were collected following allergen challenge, one from each nare.
Allergic rhinitis subjects studied using the second protocol underwent both relevant and irrelevant nasal allergen challenges at least 4 wk apart. Irrelevant allergens were selected to be allergens to which the subjects were skin test negative and had no history of clinical symptoms with natural exposure. The relevant allergen dose was the previously established provoking dose while the irrelevant allergen dose was selected at the average provoking dose for the relevant allergen challenges (1/100 dilution of the allergen concentrate). To be included, relevant allergen challenges needed to cause an increase in nasal symptoms while irrelevant challenges needed not to cause increased nasal symptoms. For each of the challenges, subjects had a baseline nasal sample obtained from one nare then immediately underwent nasal allergen challenge in the contralateral nare. Four to 8 h after challenge, subjects had an additional nasal sample obtained from the challenged nare.
Kinin challenges. Baseline nasal sample was obtained from one nare, immediately followed by instillation of the appropriate concentration of either BK or Lys-des-Arg-BK in a 100 µl spray onto the inferior turbinate of the contralateral nare. The kinin was instilled by gentle insufflation in the region of the inferior turbinate using a tuberculin syringe placed in the nare under visual inspection using a nasal speculum. A subsequent nasal sample was collected from the challenged nare 20 min after kinin was instilled. Kinin challenges were conducted at least 1 mo after allergen challenge and at least 2 wk apart. BK was obtained from PolyPeptide Laboratories (Wolfenbutel, Germany) at >98% purity as a trifluoroacetate salt reconstituted in sterile water. Lys-des-Arg-BK was obtained from Bachem (Torrance, CA) at >99% purity as a trifluoroacetate salt reconstituted in sterile water.
Sample collection
Nasal sample collection. Nasal samples were collected by gently scraping the inferior turbinate with a Rhinoprobe curette (Arlington Scientific, Springville, UT). The sample was then placed into RLT lysis buffer (Qiagen, Valencia, CA) and snap frozen. Total RNA was extracted from the samples using the RNeasy mini kits (Qiagen) according to the manufacturer’s instructions. Protein was isolated from the RNeasy Mini Spin column flow-through by TCA precipitation. An equal volume of cold 10% TCA solution was added to the flow-through and kept on ice for 20 min, followed by a 15-min microcentrifugation. Pellets were washed with 100% ice-cold ethanol and resuspended in sample buffer for immunoblotting.
Isolation of nasal epithelial cells. Nasal samples were immediately placed in 1 ml of disaggregation buffer (0.1% collagenase XI, 0.1 mg/ml DNase I, 3 mM DTT in PBS) and gently rotated for 20 min at room temperature. The nasal cells were then washed three times in cold sterile PBS containing 0.1% BSA then resuspended in 1 ml PBS containing 20% FCS. Epithelial cells were isolated from the cell suspension using the CELLection Epithelial Enrich system (Dynal Biotech, Lake Success, NY), according to the manufacturer’s instructions.
Ribonuclease protection assay (RPA)
Specific mRNA levels for the B1 receptor, B2 receptor, IL-8
gene, and L32 housekeeping gene in the nasal samples were
measured by multiprobe RPA as previously described (14).
PCR products for each of these genes were cloned into the pGEM3Z
plasmid (Promega, Madison, WI) using BamHI and
HindIII restriction sites, and the correct sequence was
confirmed on a DNA sequencer (373A Sequencer; Applied Biosystems,
Foster City, CA). Twenty micrograms of each plasmid were
linearized with the appropriate restriction enzyme and antisense RNA
probes were prepared by in vitro transcription with either T7 (B2
receptor and L32) or SP6 (B1 receptor and IL-8) RNA polymerases
(Promega) with the incorporation of
[
-32P]UTP (800 Ci/mmol; Amersham, Arlington
Heights, IL). Plasmid DNA was digested with RNase-free DNase I (10 U;
Stratagene, La Jolla, CA). The labeled riboprobes were then
gel-purified on a 6% polyacrylamide gel. Riboprobes were diluted in
hybridization buffer (Torrey Pines Biolabs, Houston, TX) to
1 x 105 cpm/µl. Total RNA was quantitated
fluorometrically using SYBR green II (Molecular Probes, Eugene, OR)
(23). Ten microliters of RNA sample (1 µg total RNA in
diethyl pyrocarbonate-treated H2O) was
heated at 95°C for 5 min then hybridized with 1 µl of labeled
riboprobe mix for 16 h at 55°C in a total volume of 20 µl
hybridization buffer. Unhybridized single-stranded RNA was digested
with 100 µl of RNase digestion mixture for 1 h at 30°C. After
the addition of 100 µl stop buffer (Torrey Pines Biolabs), the tube
was vortexed and incubated at 23°C for 10 min. The undigested RNA was
then purified by ethanol precipitation, and separated on a 6%
acrylamide/urea sequencing gel. The protected bands were visualized and
quantified by scanning the gels using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
Extracellular signal-regulated kinase (ERK) immunoblotting
Ten microliters of extracted nasal protein (0.5–1.0 µg/µl) per lane was separated by 12% SDS-PAGE then electrophoretically transferred to nitrocellulose membranes. Phosphorylated ERK was detected by immunoblotting the membrane with a mouse derived mAb specific for the phosphorylated tyrosine motif at 1/2000 dilution (Phospho-P44/42 MAP Kinase E10 Monoclonal Ab; Cell Signaling Technology, Beverly, MA) followed by HRP-conjugated goat anti-mouse IgG1 (Caltag Laboratories, Burlingame, CA). Protein bands were detected by ECL (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL). The membranes were then stripped and total ERK levels were measured by immunoblotting with a rabbit p44/42 MAPK Ab at 1/1000 dilution (Cell Signaling Technology), followed by HRP-conjugated anti-rabbit Ab (Cell Signaling Technology) and ECL (Pierce).
Cell culture
The transformed human bronchial epithelial cell line, BEAS-2B (CRL-9609; American Type Culture Collection, Manassas, VA) was grown as monolayers in 100% humidity and 5% CO2 at 37°C in the serum-free defined growth media (Clonetics, San Diego, CA). The type II pneumocyte-like human lung adenocarcinoma cell line, A549, was grown as monolayers in 100% humidity and 5% CO2 at 37°C in Ham’s F12K medium containing 2 mM L-glutamine, penicillin (100 IU/ml), streptomycin (50 µg/ml), and 10% FBS. Normal primary human bronchial epithelial cells were purchased from Clonetics, grown in serum-free defined growth media, and used on passages 2 to 4.
EMSA
Nuclear extracts were prepared by a modified method of Dignam et
al. (24) and EMSA were performed as previously described
(25). Oligonucleotides and their complementary strands for
EMSAs were from Promega. The sequence of the AP-1 oligonucleotide was
5'-CGCTTGATGAGTCAGCCGGAA-3' (E3202). Double-stranded
oligonucleotide (5 pmol) was 32P-labeled with T4
polynucleotide kinase. [
-32P]ATP (>5000
Ci/mmol) was from Amersham.
Real-time quantitative RT-PCR
Total RNA was extracted from nasal scrape samples as described above and contaminating DNA digested using a DNase-treatment kit (Qiagen). Total nasal RNA (250–500 ng) was reverse-transcribed using the Omniscript RT kit (Qiagen), and one-twentieth of the cDNA was used for subsequent PCR.
The Bio-Rad iCycler (Hercules, CA) was used for real-time quantitative PCR. The primers used were as follows: B1 receptor forward primer, 5'-CAACTGAACGTGGCAGAAATCTAC; B1 receptor reverse primer, 5'-CAAGCCCAAGACAAACACCAG; GAPDH forward primer, 5'-GGGAAGGTGAAGGTCGGAGT; GAPDH reverse primer, 5'-TCCACTTTACCAGAGTTAAAAGCAG. The following dual-labeled probes were obtained from Biosearch Technologies (Novato, CA): GAPDH, 5'-FAM-ACCAGGCGCCCAATACGACCAA-3'TAMRA; B1 receptor, 5'-FAM-TGGCCAACCTGGCAGCCTCTGA-3'BHQ-2 (black hole 2). Standards, from 10 to 0.0001 attomol of the PCR product cloned into pGEMTeasy, were run alongside the samples to generate a standard curve. All samples and standards were analyzed in triplicate. The PCR consisted of 1.5 mM Tris-HCl, 5 mM KCl, 2 mM dNTPs, 200 ng of sense and antisense primers, either 5 pmol of B1 receptor dual-labeled probe or 12 pmol of GAPDH dual-labeled probe, 4 mM Mg2+, and 1 U of AmpliTaq Gold (Applied Biosystems) in a total volume of 50 µl. The reaction conditions were 95°C for 10 min followed by 50 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C. The starting amount of cDNA in each sample was calculated using the iCycler software package (Bio-Rad).
Statistics
All analyses were performed using nonparametric techniques. Nonpaired comparisons were analyzed using the Mann-Whitney U test and paired comparisons were analyzed using the Wilcoxon signed rank test. Correlation between two variables was assessed using the Spearman rank correlation test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nasal samples were obtained using the Rhinoprobe curette from 34
subjects with active allergic rhinitis and 17 normal subjects.
Steady-state mRNA levels of the B1 receptor as well as the B2 receptor,
IL-8 gene, and L32 ribosomal housekeeping gene were measured by
multiprobe RPA. In all cases, BK receptor and IL-8 mRNA levels were
normalized to the level of L32 mRNA expression based on the density of
the respective bands as determined by phosphorimaging. Fig. 1
shows a representative pattern from
four allergic rhinitis subjects and two normal controls. Strong
protected B1 receptor mRNA bands were detected in two of the allergic
rhinitis samples with a third allergic rhinitis subject showing a weak
but clear band. B1 receptor protected bands were not seen in the normal
subject samples. In contrast, all six samples (allergic rhinitis and
normal) showed clear B2 receptor bands.
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summarizes
the B1 receptor mRNA expression in all 51 subjects. Interestingly 14 of
17 (82.4%) normal subjects did not express detectable B1 receptor
mRNA; however, even among the allergic rhinitis subjects, 13 of 34
(38.2%) also did not express detectable B1 receptor mRNA. To assess
the repeatability of the multiprobe RPA, several nasal scrape samples
from allergic rhinitis subjects were pooled, then analyzed, in five
replicates. The mean B1 receptor/L32 mRNA ratio for the five replicates
was 33.04% with a standard error of 2.32%.
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To assess whether allergic inflammation could directly increase B1 receptor expression, we measured B1 receptor mRNA in nasal tissue samples before and after in vivo nasal allergen challenge. Seven allergic rhinitis subjects with mild or quiescent symptoms at baseline were challenged with relevant allergen and experienced a spectrum of typical symptoms including itch, congestion, sneeze, rhinorrhea, sore throat, and ocular watering and itch. Nasal samples were obtained 1 wk before allergen challenge (baseline) then 8 and 24 h after allergen challenge in six subjects or 4 and 8 h after allergen challenge in one subject. B1 receptor and L32 mRNA levels were measured by RPA, and the B1 receptor expression was normalized to L32.
Six of seven allergen-challenged allergic rhinitis subjects
demonstrated increased B1 receptor mRNA following allergen challenge.
Fig. 3 shows the changes in B1 receptor
mRNA expression at the times following challenge compared with the
baseline values. The peak increase in B1 receptor mRNA occurred at
8 h in four of the seven subjects. Two subjects showed peak B1
receptor mRNA at 24 h, and one subject did not show any change.
Comparing each subjects’ maximal postchallenge level to the baseline
level, allergen challenge resulted in a statistically significant
increase in B1 receptor mRNA expression (p <
0.05; Wilcoxon signed rank test). As an additional control, a single
normal subject was challenged with grass allergen and measurements of
B1 receptor message analyzed at 4 and 8 h time points by RPA. No
detectable message was found in the baseline or postchallenge samples
(data not shown).
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To determine whether nasal epithelial cells expressed B1 receptor mRNA, we isolated nasal epithelial cells from the nasal scrape samples in two normal and two allergic rhinitis subjects. Total RNA was isolated from the epithelial cells and the ratio of B1 receptor to GAPDH mRNA was measured by real-time quantitative RT-PCR. Detectable B1 receptor was found in all four subjects, and the mean B1 receptor/GAPDH mRNA ratio in the two normal subjects was virtually identical to the ratio found in total nasal scrape samples (0.0040 and 0.0057). However, one of the allergic rhinitis subjects showed a >10-fold higher level of B1 receptor compared with the normal subjects.
Activation of ERK in cultured airway epithelial cells following kinin challenge
BK has been previously reported to activate the MAPK ERK in a
variety of cell types (26, 27). Therefore, we examined the
ability of BK and Lys-des-Arg to activate ERK-1 and ERK-2 in human
airway epithelial cells. The transformed human bronchial epithelial
cell line, BEAS-2B, constitutively expresses both B2 and B1 receptors
(data not shown). Following stimulation with BK, an increase in
phosphorylated ERK-1 and ERK-2 was seen beginning by 7.5 min (the
earliest time point sampled) and continuing for at least 22.5 min (Fig. 4). Stimulation of BEAS-2B cells with
Lys-des-Arg-BK also increased phospho-ERK1/2, with the increase first
seen at 15 min and continuing for at least 22.5 min (Fig. 4).
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). However, in marked
contrast to the BEAS-2B cells, primary human bronchial epithelial cells
do not activate ERK in response to Lys-des-Arg-BK (Fig. 5). These
results show that the B1 receptor is required in order for
Lys-des-Arg-BK to activate ERK.
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To assess whether the increased B1 receptor mRNA found in nasal samples from allergic rhinitis subjects was associated with expression of functional receptors, we performed nasal challenges with BK or Lys-des-Arg-BK in allergic rhinitis and normal subjects then measured in vivo activation of ERK in baseline and 20 min postchallenge nasal samples. Both normal and allergic subjects reported transient nasal burning, sore throat, and congestion in response to the BK challenge, usually maximal at 1–5 min with resolution by 20 min. No subjects experienced any symptoms in response to Lys-des-Arg-BK challenge.
As would be expected for the constitutively expressed B2 receptor,
instillation of 200 µg BK resulted in activation of ERK in both
normal and allergic rhinitis subjects (Fig. 6A). In contrast, instillation
of 500 or 200 µg of Lys-des-Arg-BK activated ERK in allergic rhinitis
subjects while instillation of 500 µg Lys-des-Arg-BK did not activate
ERK in a normal subject (Fig. 6B). Summary data from the in
vivo kinin challenges are summarized in Fig. 7. Values are expressed as the fold
change in the ratio of the phospho-ERK to total ERK in the
postchallenge sample vs the matched baseline sample. All seven of the
allergic rhinitis subjects challenged with Lys-des-Arg-BK demonstrated
an increase in phospho-ERK (four challenged with 500 µg and three
challenged with 200 µg). In contrast, six of seven normal controls
challenged with Lys-des-Arg-BK (five challenged with 500 µg and one
challenged with 200 µg) showed no increase in phospho-ERK. A single
normal subject, challenged with 500 µg Lys-des-Arg-BK, showed a small
increase in phospho-ERK. The difference in response to Lys-des-Arg-BK
challenge between the allergic rhinitis and normal subjects was
statistically significant (p < 0.005;
Mann-Whitney U test). Fig. 7 also shows that all eight
allergic rhinitis and four normal subjects challenged with BK showed
increased activation of ERK in the 20-min sample compared with the
baseline sample. These results are compatible with constitutive nasal
expression of B2 receptors in both subject groups with expression of
functional B1 receptors only in the allergic rhinitis subjects.
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BK-mediated activation of ERK through the Ras-Raf pathway has been
associated with activation of the transcription factor AP-1
(27). Therefore, we assessed the effects of BK and
Lys-des-Arg-BK on transcription factor activation in A549 cells, a
transformed type II pneumocyte cell line (Fig. 8). As we previously reported, BK
stimulated activation of transcription factor NF-
B (Fig. 8A, lane 4), and this could be blocked by
preincubation with a B2 receptor antagonist (Fig. 8A,
lane 10). In contrast, Lys-des-Arg-BK did not stimulate
NF-B activation (Fig. 8A, lane 3). However,
both BK and Lys-des-Arg-BK stimulated AP-1 activation (Fig. 8B, lanes 3 and 4). BK-stimulated AP-1
activation was blocked by a B2 receptor antagonist (Fig. 8B,
lane 10), but not a B1 receptor antagonist (Fig. 8B, lane 7); while Lys-des-Arg-BK-stimulated AP-1
activation was blocked by a B1 receptor antagonist (Fig. 8B,
lane 6), but not a B2 receptor antagonist (Fig. 8B, lane 9). Lys-des-Arg-BK also activated AP-1
in BEAS-2B bronchial epithelial cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We report the novel observation that kinin B1 receptors are induced during allergic airway inflammation in human subjects. Using multiprobe RPA to measure expression in nasal samples, we found a statistically significant increased level of B1 receptor mRNA in 34 allergic rhinitis subjects compared with 17 normal controls. B1 receptor mRNA was not detectable in 82% of normal subjects (14 or 17), in keeping with animal and human cell line data indicating that the B1 receptor is usually absent in normal tissue and only expressed in response to pathophysiologic stimuli (17). Within the group of allergic subjects, there was significant variability in the level of message expression, with 38% of allergic rhinitis subjects having no detectable receptor. B1 receptor mRNA expression was also measured by quantitative real-time RT-PCR. Using this more sensitive technique, we could consistently detect low level expression of B1 receptor mRNA in the normal subjects, however the allergic rhinitis subjects showed significantly higher levels of expression. Using multiprobe RPA and real-time quantitative RT-PCR, we also demonstrated that relevant allergen challenge significantly increases nasal B1 receptor mRNA expression in subjects with mild or quiescent allergic rhinitis. Although a certain amount of variability was evident with respect to B1 receptor expression, there was clear evidence of elevated B1 receptor mRNA expression in allergic rhinitis subjects which was further increased in the majority of subjects by allergen challenge.
Little is known regarding the potential consequences of agonist binding to the expressed B1 receptors in the airway. The lack of a phosphorylation motif in its C-terminal intracellular domain has been reported to cause the B1 receptor to be only minimally internalized and desensitized, thus allowing for protracted activity (31). Studies in transiently transfected human embryonic kidney 293 cells have also shown that the B1 receptor exhibits a high degree of constitutive activity even in the absence of ligand (32). BK has been reported to activate the MAPK ERK in a variety of cells (27, 33). Although des-Arg-BK has also been reported to activate ERK in human embryonic kidney 293 cells overexpressing B1 receptors (34), the significance of this observation is uncertain because des-Arg-BK is not an effective agonist for the human B1 receptor (11, 12).
Using cultured human airway epithelial cells, we demonstrated that engagement of either the B1 or B2 receptor by its appropriate agonist resulted in rapid phosphorylation of ERK-1/ERK-2. BK was used as the B2 receptor agonist and Lys-des-Arg-BK was used as the B1 agonist. The specificity of their agonist activity at the concentrations used have been carefully documented. BK and Lys-BK (kallidin) are equally effective agonists for the B2 receptor; however, Lys-BK, but not BK, shows some agonist activity for the B1 receptor (11, 12). Their carboxypeptidase metabolites (des-Arg-BK and Lys-des-Arg-BK) have very different B1 receptor agonist activities; Lys-des-Arg-BK is a potent agonist for the human B1 receptor, while des-Arg-BK is at least 1000-fold less potent (11, 12).
We then used nasal kinin challenges to examine in vivo activation of ERK in nasal cells. Seven allergic rhinitis subjects and seven normal controls were challenged with Lys-des-Arg-BK. Postchallenge samples were collected at 20 min based on the in vitro response to agonists in BEAS-2B cells. All seven allergic rhinitis subjects demonstrated clear activation of ERK following challenge with either 500 or 200 µg of Lys-des-Arg-BK. The seven normal subjects were all challenged with 500 µg of Lys-des-Arg-BK, however only a single normal subject showed minimal activation of ERK. In contrast, BK nasal challenge activated ERK in both allergic rhinitis and normal subjects. These results both confirm the expression results discussed above and demonstrate that the nasal B1 receptor mRNA found in allergic rhinitis subjects is associated with expression of functional B1 receptors.
Activation of ERK has been linked to activation of the transcription factor AP-1 (27, 35), and previous studies have suggested that signaling through the B1 receptor may contribute to the regulation of gene expression (36, 37, 38). We demonstrated that Lys-des-Arg-BK could activate AP-1 in transformed human respiratory epithelial cells (A549 and BEAS-2B). AP-1 has an array of associations with potentially important regulatory pathways involved in allergic inflammation of the airways, including stimulation of cytokine and chemokine transcription and functioning as an effector of growth factor signaling required for normal cell cycle progression and in the apoptotic response (39, 40, 41). Preliminary experiments in our laboratory suggest, moreover, that Lys-des-Arg-BK stimulates increased expression of chemokine receptors in cultured airway epithelial cells (data not shown). The timing of B1 receptor expression following allergen challenge suggests the possibility that B1 receptors may contribute to the phenomena of the late-phase response, which has been clearly linked to chronic airway inflammation (42). Additional studies will be required to define the potential effects and role of the B1 receptor in airway gene expression during allergic inflammation.
In summary, we have demonstrated the up-regulation of B1 receptors in
naturally occurring allergic rhinitis and in response to challenge with
relevant allergen. Evidence is also presented confirming the functional
signaling of the B1 receptors in response to ligand exposure in
allergic, but not normal, subjects. Finally, we have provided evidence
that engagement of the B1 receptor with its ligand leads to activation
of AP-1, a transcription factor that regulates both basal and inducible
transcription of many genes involved in cellular proliferation and
differentiation. The ability of the B1 receptor to activate AP-1
represents a novel and potentially important functional consequence of
receptor up-regulation. We hypothesize that the kinin system
contributes to the development of airway inflammation initially through
the B2 receptor, with subsequent up-regulation of the B1 receptor.
Therefore, generation of kinins in the airway will activate both
NF-B and AP-1 which act together to stimulate transcription of many
genes involved in the inflammatory response. The B1 receptor is
postulated through these avenues to play a role in sustaining and
amplifying chronic inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce L. Zuraw, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: zuraw{at}scripps.edu
3 Abbreviations used in this paper: BK, bradykinin; MAPK, mitogen-activated protein kinase; RPA, ribonuclease protection assay; ERK, extracellular signal-regulated kinase.
Received for publication July 17, 2001. Accepted for publication June 5, 2002.
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) subjects. The mean ± SEM values
are indicated by the box shown for each group.
70% confluency
then stimulated with either medium alone (control), LPS (100 ng/ml), BK
(10 nM), or Lys-des-Arg-BK (10 nM). At the times indicated, the cells
were washed then lysed. The amount of phospho-ERK and total ERK in each
sample was determined by immunoblotting as described in
Materials and Methods. The autoradiographs of the
membranes are shown with the phospho-ERK on the top (pp44
and pp42) and the corresponding total ERK on the bottom (p44
and p42).
