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*
Department of Microbiology, University of Western Australia, Nedlands, Western Australia;
TVW Telethon Institute for Child Health Research, West Perth, Western Australia; and
Department of Medicine, University of Western Australia, Queen Elizabeth II Medical Center, Nedlands, Western Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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10 ng/ml.
Cytokine release was initiated by 1 to 2 h of protease exposure,
although maximum concentrations were detected only after a 24-h
incubation. IL-6, but not IL-8 and GM-CSF, was shown to be degraded by
both proteases at concentrations of >2 µg/ml. The proteases also
stimulated changes in the intracellular calcium ion concentration. All
mite protease-induced phenomena were inhibited using appropriate
protease inhibitors. These results suggest that the proteolytic
activity of an allergen may stimulate the release of proinflammatory
cytokines from human bronchial epithelium. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human respiratory epithelium is a critical component of the innate
immune system, acting as both a physical barrier to the environment and
a modulator of local airway inflammation because of its capacity to
synthesize a variety of mediators including cytokines, prostanoids,
leukotrienes, nitric oxide, antioxidants, and complement components
(1, 2, 3). Each of these mediators possesses the capacity to
influence the activation and migration of inflammatory cells, modulate
smooth muscle tone, or initiate mucosal repair in respiratory disease.
In asthma, the respiratory epithelium releases cytokines such as IL-1,
IL-6, granulocyte-macrophage
(GM)-CSF,4 TNF-
, and
TGF-ß, as well as chemokines such as IL-8, macrophage inflammatory
protein-1, and RANTES (2, 4, 5); such mediators are
likely to be particularly important in the pathogenesis of this
disease.
The precise mechanism(s) underlying the release of cytokines from
respiratory epithelium is unclear, but a variety of factors have been
identified. These factors include eosinophil granule proteins, ozone,
isocyanates, other cytokines such as IL-1 and TNF-, viruses, and
bacteria (2, 6, 7, 8). More recently, studies have shown that endogenous
proteases from neutrophils (9), mast cells (10), and cytotoxic T cells
(11) also stimulate the release of cytokines from this tissue.
Interestingly, the airway epithelium is also exposed to exogenous
proteases in the form of allergens, particularly those originating from
the house dust mites Dermatophagoides pteronyssinus and
Dermatophagoides farinae. These allergens include the
group 1 cysteine proteases (12), the group 3 trypsins (13), the group 6
chymotrypsins (14), and the recently identified group 9 collagenolytic
serine proteases (15).
Interestingly, we have shown that these proteases possess the capacity to modulate respiratory mucosal permeability and vascular permeability (16, 17); this observation suggests that allergens possess the capacity to modulate epithelial function. On this basis, we have undertaken studies to determine whether mite allergens also initiate cytokine release from respiratory epithelium. The data show that the cysteine protease Der p 1 and the serine protease Der p 9 allergens from D. pteronyssinus enhance the release of cytokines from both primary cultures of human bronchial epithelium and the bronchial epithelial cell line BEAS-2B. This release was dependent upon protease activity per se and was accompanied by changes in the flux of intracellular calcium ions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extracts of D. pteronyssinus were prepared from fecally enriched spent growth medium as described previously (13). General chemicals were purchased from BDH (Kilsyth, Australia), and synthetic protease substrates and other reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated.
Purification of mite protease allergens
Der p 1 and Der p 9 were isolated as described previously (15). In brief, Der p 1 was isolated by mAb affinity chromatography, and Der p 9 was isolated using a combination of ion exchange chromatography and soybean trypsin inhibitor (SBTI) affinity chromatography. The proteolytic activities of Der p 1 and Der p 9 were determined as described previously (15) using the insoluble collagen substrate, Azocoll (Calbiochem, Alexandria, Australia) and the synthetic substrate succinyl-alanyl-alanyl-prolyl-phenylalanyl-p-nitroanilide, respectively.
Primary epithelial cell culture
Human bronchi were obtained from thoracotomy specimens as described previously (18) and were dissected to be free of all visible blood vessels and parenchyma; primary epithelial cell cultures were derived from explants. Briefly, tissue pieces were stripped from the airway wall and cut into 2-mm2 pieces that were then orientated epithelial surface uppermost on plastic culture dishes; the dishes had been previously rinsed with serum-free tissue culture medium consisting of LHC basal medium (Biofluids, Rockville, MD) supplemented with insulin, hydrocortisone, epidermal growth factor, bovine hypothalamus extract, cholera toxin, transferrin, and retinol acetate as described previously. The tissue explants were allowed to adhere onto rat type I collagen-coated (30 µg/ml) plastic culture dishes at 37°C for 16 h after which supplemented basal medium was added gently to avoid dislodging the tissue. The medium was changed at 2- to 3-day intervals until the outgrowths had reached the edge of the dish (12–20 days). The explants were removed, and cells were cultured for an additional 24 h before use in basal medium that was devoid of additives.
Characterization of epithelial cells
The purity of the primary epithelial cell cultures was confirmed by staining with anti-cytokeratin Abs. Cells were fixed in methanol for 10 min, rinsed with 0.01 M of PBS (pH 7.2), and stained with an anti-cytokeratin mAb (clone LP34; Dako, Botany, Australia) at a dilution of 1/50 in PBS for 30 min at room temperature. The slides were washed in PBS, and rabbit alkaline phosphatase-conjugated anti-mouse IgG was applied for 30 min at room temperature; the slides were subsequently rinsed, and the reaction was visualized using fast red substrate. Only epithelial cell cultures showing <3% contamination with fibroblasts were studied.
Human bronchial epithelial cell line culture (BEAS-2B)
BEAS-2B, an SV40-transformed human bronchial epithelial cell line, was kindly provided by Prof. C. C. Harris (National Institutes of Health, Bethesda, MD) and maintained at 37°C/5% CO2 on collagen-coated plastic culture dishes in supplemented LHC medium (19). Cells were passaged by incubating the monolayer with prewarmed 0.05% (w/v) trypsin (Life Technologies, Melbourne, Australia)/0.02% (w/v) EDTA solution for 10 min at 37°C. Basal medium containing 20% (w/v) SBTI was added to the cell suspension, and the cells were pelleted by centrifugation (1200 rpm for 5 min). Following centrifugation, the cells were resuspended in 1 ml of basal medium, counted by trypan blue exclusion to determine cell number and viability, and plated at a concentration of 1 x 105 cells/ml in 200-µl volumes in 24-well plates.
Stimulation of epithelial cells in culture
Epithelial cells were washed and cultured in basal medium at
24 h before stimulation, and 5 x 106
confluent cells were incubated with either Der p 1 or Der p 9, Der p 1
or Der p 9 plus appropriate inhibitors (E-64, SBTI, and
1-antiprotease (1-AP)), protease
inhibitor alone, heat-inactivated protease (65°C for 15 min),
polymyxin B, rIL-1, LPS, or basal medium. Experiments were also
performed using the bovine proteases thrombin (Rhóne-Poulenec
Rorer, Victoria, Australia) and trypsin. All additives were prepared in
prewarmed (37°C) basal medium, and inhibitors were incubated with
protease for 10 min at 37°C before being added to cell cultures.
Supernatant or cells from protease-treated epithelium and nontreated
controls were obtained and tested for the presence of various cytokines
or cytokine mRNA as described below. The possible cytotoxic effects of
various additives were assessed by trypan blue exclusion.
Cytokine assays
The presence of GM-CSF in the supernatants was determined by bioassay using the GM-CSF/IL-3-dependent cell line FDC-P1 (1 x 104 cells/well), which was kindly provided by Professor P. G. Holt (TVW Telethon Institute for Child Health). The proliferation of cells in response to the cytokine was determined by the incorporation of [3H]thymidine (Amersham, Sydney, Australia), which was added at 1 µCi/well for the last 4 h of the 3-day incubation period. Cells were harvested on fiberglass filters, and the radioactivity was measured by scintillation counting. The amount of GM-CSF was determined using rGM-CSF, which was kindly provided by Dr. A. S. McWilliam (TVW Telethon Institute for Child Health). Specificity was confirmed using a blocking goat anti-human GM-CSF (R&D Systems, Minneapolis, MN). Exposing the FDC-P1 cells to protease alone did not elicit proliferation. The concentrations of IL-6 and IL-8 were measured by a capture ELISA. For the IL-6 ELISA, the capture Ab used (1 µg/ml) was a goat anti-human IL-6 polyclonal Ab (R&D Systems), and the detection Ab used was a mouse anti-human IL-6 mAb (BioSource International, Camarillo, CA) at 1 µg/ml; the standard used was human rIL-6, which was kindly provided by Dr. R. J. Simpson (Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research and the Walter and Elisa Hall Institute for Medical Research, Parkville, Australia). For the IL-8 ELISA, the capture Ab used (2 µg/ml) was a rabbit anti-human IL-8 polyclonal Ab (R&D Systems), and the detection Ab used was a mouse monoclonal anti-IL-8 at 2 µg/ml; the standard used was human rIL-8 (BioSource International). Horseradish peroxidase-conjugated sheep anti-mouse Ig (Amersham) and tetramethylbenzidine substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were used to detect secondary Ab binding in both assays. The enzyme reactions were stopped with 1 M sulfuric acid, and ODs were determined at 450 nm. The assays were log-linear from 0.1 to 10 ng/ml of cytokine. All data were expressed as nanograms or picograms of cytokine released per 106 cells, and the significance of the differences in mean responses was determined using the Student t test.
Northern blot analyses
RNA was extracted from mite protease-treated or nontreated control epithelial cell cultures with RNAzol (Bresatec, Adelaide, Australia), electrophoresed on 1% (w/v) agarose gels containing 37% (v/v) formaldehyde before transfer onto a nylon membrane (Hybond N+, Amersham), and probed for IL-6 using 1000-bp BamHI fragments that were kindly provided by DNAX (Palo Alto, CA). Control experiments were performed using a glyceraldehyde phosphate-3-dehydrogenase (GAPDH) probe that was kindly provided by Associate Prof. U. Kees (TVW Telethon Institute for Child Health). Each cDNA fragment was labeled with [32P]deoxyATP by random priming, and hybridization was performed sequentially at 65°C. The intensities of the signals were determined by densitometric quantification and normalized to the responses that were obtained with the GAPDH probe.
Measurement of cytosolic free calcium (Ca2+)
Intracellular Ca2+ signaling following stimulation with dust mite proteases was determined in three separate experiments using real-time fluorescence measurements at the single-cell level. BEAS-2B cells were grown to confluence on optical grade circular glass coverslips (22-mm2 and 0.14–0.16-µm thickness) and loaded intracellularly with 1 µM Indo-1/AM (Calbiochem) in loading buffer (145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 2.5 mM CaCl2, 10 mM glucose, 25 mM HEPES, and 0.5 mM MgSO4) for 30 min at 37°C. Cells were gently washed in loading buffer, and real-time fluorescence measurements were obtained digitally using an interactive laser cytometer (ACAS 470, Bio-Rad, Regents Park, Australia). Proteases were added to the cells after a 25-s scan of a group of 10 to 20 adherent BEAS-2B cells, and scanning was continued for another 100 s. Changes in cytosolic-free calcium were expressed as the ratio of the fluorescence obtained using detector 1 (free Indo-1, 485 nm) to that obtained with detector 2 (Indo-1: Ca2+ complex, 405 nm).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments were performed to determine whether the mite
proteases caused cytokine release from BEAS-2B cells. These studies
showed (Fig. 1
) that both Der p 1 and Der
p 9 caused the release of GM-CSF, IL-6, and IL-8 in a dose-dependent
manner after 24 h of exposure. Statistically significant cytokine
responses above background were observed with 10 ng/ml of either
protease for all cytokines with the exception of Der p 1-induced GM-CSF
responses, which required a concentration of 50 ng/ml. For GM-CSF,
maximum responses were observed at 2 µg/ml, whereas maximum
responses were not achieved with either IL-6 and IL-8 at the maximum
concentration tested, namely 4 µg/ml. With IL-6, a diminution in
cytokine response was observed after stimulation with both proteases
but was most marked with Der p 9. Subsequent studies (Table I) showed that IL-6 was susceptible to
proteolysis by both Der p 1 and Der p 9. There was a significant trend
for higher concentrations of IL-6 to be produced by Der p 9 than by Der
p 1, but this trend was reversed for IL-8 (Fig. 1). Table II shows the mean concentrations of the
IL-6, IL-8, and GM-CSF released by the two mite proteases at a
concentration of 100 ng/ml compared with those released using the
mammalian proteases trypsin and thrombin. It is evident that, with the
exception of the trypsin-induced GM-CSF release, all proteases induced
significant cytokine release.
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) indicated that the earliest timepoint
at which the statistically significant mean concentrations of the three
cytokines studied could be detected for the BEAS-2B cell line was at
8 h after treatment with both proteases. Similar results were
obtained for the GM-CSF and IL-6 released from primary cell cultures
after treatment with Der p 1 and Der p 9; statistically significant
mean differences in IL-8 production were not observed, although there
was a trend for higher concentrations to be produced. There was a
tendency for the primary cultures to produce greater mean GM-CSF
release with both proteases, although this tendency was not
significant. For the BEAS-2B cell line, significantly more IL-6
was produced after 8 h of incubation with Der p 9 than Der p 1
(p < 0.02). The effects of varying the period
of protease exposure on the accumulated release of IL-6 and IL-8 at
24 h were subsequently determined using the BEAS-2B cell line.
These studies showed (Fig. 3) that the
release of both cytokines was evident after a 30-min exposure and
corresponded to 20 to 60% of the maximum amount released. By 1
h, the amount of IL-6 released by Der p 9 was maximal, and maximal IL-6
release was achieved after 2 h with Der p 1. Maximal IL-8
release with both proteases was achieved by a 2-h exposure. Trypan blue
dye exclusion studies showed that the effects that were observed at the
protease concentrations used were not related to cell death; these
findings are in accord with those from previous studies using mite
enzymes (16).
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Northern blot analyses demonstrated that IL-6 mRNA transcripts
were undetectable in resting BEAS-2B cells (Fig. 4); however, cytokine mRNA levels
increased in cells treated with mite proteases, reaching a maximum at
2 h postexposure and subsiding after 8 h. This modulation was
not observed with GAPDH (Fig. 4).
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The mechanism of cytokine induction from human bronchial
epithelial cells was investigated by preincubating 100 ng/ml of each
protease allergen with the appropriate inhibitors. For the serine
protease Der p 9, this incubation included SBTI and
1-AP; for the cysteine protease Der p 1, the incubation
included E-64. The effect of 1-AP on Der p 1 was also
assessed, given recent claims that protease activity could be inhibited
by this protein (20). The data obtained indicated that IL-6 and
IL-8 release (Fig. 5) by both Der p 1 and
Der p 9 was abolished when protease activity per se was inhibited, and
that 1-AP had no effect on the cytokine-inducing effects
of Der p 1. Similarly, IL-6 mRNA expression was ablated when both
proteases were inhibited (Fig. 4
). As some protease inhibitors have
been shown to block the activation of the transcription factor NF-
B
as well as subsequent cytokine gene activation (21), the effect of SBTI
on IL-1ß-induced cytokine release from BEAS-2B cells was
investigated. These studies showed that this inhibitor did not affect
release at the concentrations used. The cytokine-releasing effects of
the mite proteases did not appear to be associated with LPS, since
adding the endotoxin antagonist polymyxin B did not affect cytokine
release; heat inactivation of protease activity did affect the release
of cytokines however (Fig. 5).
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The effects of Der p 1 and Der p 9 on intracellular calcium
(Ca2+) in BEAS-2B cells that had been loaded
intracellularly with the Ca2+-sensitive fluorescent dye
Indo-1 were determined using cell monolayers grown on glass coverslips.
Both mite proteases induced changes in (Ca2+)i in 90% of
the cells studied. Figure 6 shows the
representative data that were obtained with cells that had been treated
with 1 µg/ml of Der p 1 and Der p 9; changes occurred within 10
s of protease addition. There was a slight variation in response from
cell to cell in terms of response time and the intensity of response,
particularly with the cells at the edge of the coverslip, which
responded more quickly. The increases in (Ca2+)i that were
induced by Der p 9 and Der p 1 were inhibited by SBTI and E-64,
respectively. Preincubating BEAS-2B cells with Der p 1 had little
influence on the capacity of the cells to respond to a subsequent
challenge with Der p 9. However, preincubating the cells with bovine
trypsin suggested a diminution in response to a subsequent challenge
with Der p 9.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The release of both IL-6 and IL-8 by Der p 1 and Der p 9 was found to
be dependent upon biochemical activity, since both cytokines were not
detected when the proteases were inhibited by the appropriate
inhibitors. In this regard, the cytokine-releasing properties of Der p
1 were not affected by 1-AP. These data are consistent
with those indicating that this enzyme is a true cysteine protease (12, 24, 25) rather than a cysteine-serine protease (20). Cytokine release
was not related to LPS contamination and did not appear to be
associated with cell death. The responses that were obtained with the
cysteine protease allergen Der p 1 and the serine protease allergen Der
p 9 were similar, although the latter tended to be more effective than
Der p 1 at stimulating IL-6. However, the situation was reversed for
IL-8 responses, suggesting that the two proteases may be acting
on different targets, as their proteolytic specificities are likely to
be distinct. Our data also showed that IL-6, in contrast to IL-8
and GM-CSF, was susceptible to proteolytic cleavage; this finding is in
accord with previous observations (26, 27).
These studies show that enzymatically active allergens produce cytokine
responses in bronchial epithelial cells in vitro, but it is more
difficult to assess whether mite protease exposure in vivo evokes
similar responses or whether, if similar effects occur, the
concentrations of the cytokines released are of physiologic
significance. However, it is clear that the cytokine concentrations
observed in this study are likely to be biologically active given a
variety of in vitro data (28, 29); it is also apparent that the
severity of asthma as well as respiratory symptomology (coughing,
wheezing, and breathlessness) correlate with Der p 1 and general
protease concentrations in dust, respectively (30, 31, 32). Similarly, it
is not clear whether exogenous protease activity is inhibited by one or
more of the inhibitors that are known to be present in the lung given
the observation that Der p 9 may be inhibited by 1-AP
and also our unpublished observations showing that Der p 1 may
be inhibited by cystatin C. In this regard, it has been shown
that Der p 1 cleaves 1-AP in vitro (33) in a manner that
is similar to that reported for papain; this finding indicates that the
lung protease inhibitor screen may be compromised on allergen entry.
Finally, although the precise concentration of mite allergen that is
deposited on respiratory epithelium has yet to be determined, studies
have shown that the concentration of Der p 1 that is required to elicit
cytokine release in our studies is within an order of magnitude of that
demonstrated in concentrated bronchoalveolar fluid (range =
<1–10 ng/ml) obtained from individuals exposed to mite allergen (34)
overnight; in addition, this required concentration is much less than
that which was shown to be present in mite fecal pellets (>1 mg/ml)
(35), suggesting that the consequences of mite allergen exposure that
have been described are feasible.
Although the mechanism(s) underlying our findings have yet to be determined, it is unlikely that they are related to a simple proteolytic activation of cell surface-bound cytokine precursors given the up-regulation of cytokine mRNA. It is more likely that the effects observed are mediated by receptors on the epithelial cell surface. In this regard, recent studies have revealed the existence of a group of G protein-coupled receptors on a variety of cells that are activated not only by endogenous enzymes with specificities similar to those from the mite but also at similar concentrations to those reported here. This group includes receptors that are specific for thrombin and trypsin, designated proteinase-activated receptors (PARs) (36, 37, 38, 39). Thus far, four PARs have been described (39, 40), and activation is dependent upon the cleavage of the N-terminal peptide of each receptor in each case; this cleavage allows the newly created N terminus (designated as a tethered ligand) to interact with residues within the remaining receptor, resulting in activation (41). Proteases interact with these receptors to up-regulate a range of cellular functions, including cytokine secretion, cellular proliferation, and the regulation of vascular tone. In addition, the activation of those receptors is accompanied by the mobilization of cytoplasmic Ca2+ from internal stores (41); in this regard, preliminary studies indicate that mite protease-induced cytokine release was shown to behave in a fashion that was similar to and consistent with a role for PARs (36, 37, 38, 39). If receptors are involved in mite allergen-induced epithelial cell physiology, the findings that the response obtained with Der p 9 was not significantly influenced by prior exposure to Der p 1 and that a complete ablation of the response obtained with Der p 9 after treatment with trypsin was not observed suggest that there may be several types of protease receptors that are involved that warrant further study.
In conclusion, the studies described in this report suggest that the deposition of mite proteases on respiratory mucosa may induce localized inflammatory foci; these foci have the potential to influence a variety of immunologic processes in a manner similar to that postulated for agents such as viruses or airborne pollutants. For example, the release of cytokines such as GM-CSF, IL-6, and IL-8 from epithelium may have an impact on immunogenicity because of the effects of these cytokines on dendritic cell growth, MHC class II expression, airway macrophage survival, B cell differentiation, and the chemoattraction of neutrophils and eosinophils; IL-6 may also help drive naive CD4 positive T cells to the effector Th2 type (42). In addition, a variety of studies indicate that the proteolytic properties of mite allergens may also influence anaphylatoxin production, activate fibrinolysin, and cleave CD23 and CD25 on B cell surfaces (16, 17, 20, 28, 43, 44, 45). Such properties raise the possibility that along with size and solubility, the enzymatic activities of allergens are important determinants of the outcome of exposure to a foreign protein.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037.
3 Address correspondence and reprint requests to Dr. Geoffrey A. Stewart, Department of Microbiology, University of Western Australia, Nedlands, Western Australia 6907.
4 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; SBTI, soybean trypsin inhibitor; PAR, proteinase-activated receptor; 1-AP, 1-antiprotease; GAPDH, glyceraldehyde phosphate-3-dehydrogenase.
Received for publication October 7, 1997. Accepted for publication May 26, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B- is necessary for activation of transcription factor NF-B. Nature 365:182.[Medline]
B activation through interference with IB function in asthmatic bronchial epithelial cells. Biochem. Biophys. Res. Commun. 236:522.[Medline]
. Immunology 77:330.[Medline]
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Y. Sakata, K. Arima, T. Takai, W. Sakurai, K. Masumoto, N. Yuyama, Y. Suminami, F. Kishi, T. Yamashita, T. Kato, et al. The Squamous Cell Carcinoma Antigen 2 Inhibits the Cysteine Proteinase Activity of a Major Mite Allergen, Der p 1 J. Biol. Chem., February 13, 2004; 279(7): 5081 - 5087. [Abstract] [Full Text] [PDF]
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N. Asokananthan, P. T. Graham, D. J. Stewart, A. J. Bakker, K. A. Eidne, P. J. Thompson, and G. A. Stewart House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1 J. Immunol., October 15, 2002; 169(8): 4572 - 4578. [Abstract] [Full Text] [PDF]
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 J. Li, S. Kartha, S. Iasvovskaia, A. Tan, R. K. Bhat, J. M. Manaligod, K. Page, A. R. Brasier, and M. B. Hershenson Regulation of human airway epithelial cell IL-8 expression by MAP kinases Am J Physiol Lung Cell Mol Physiol, October 1, 2002; 283(4): L690 - L699. [Abstract] [Full Text] [PDF]
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J. L. Lordan, F. Bucchieri, A. Richter, A. Konstantinidis, J. W. Holloway, M. Thornber, S. M. Puddicombe, D. Buchanan, S. J. Wilson, R. Djukanovic, et al. Cooperative Effects of Th2 Cytokines and Allergen on Normal and Asthmatic Bronchial Epithelial Cells J. Immunol., July 1, 2002; 169(1): 407 - 414. [Abstract] [Full Text] [PDF]
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N. Asokananthan, P. T. Graham, J. Fink, D. A. Knight, A. J. Bakker, A. S. McWilliam, P. J. Thompson, and G. A. Stewart Activation of Protease-Activated Receptor (PAR)-1, PAR-2, and PAR-4 Stimulates IL-6, IL-8, and Prostaglandin E2 Release from Human Respiratory Epithelial Cells J. Immunol., April 1, 2002; 168(7): 3577 - 3585. [Abstract] [Full Text] [PDF]
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A. POMES, M. D. CHAPMAN, L. D. VAILES, T. L. BLUNDELL, and V. DHANARAJ Cockroach Allergen Bla g 2 . Structure, Function, and Implications for Allergic Sensitization Am. J. Respir. Crit. Care Med., February 1, 2002; 165(3): 391 - 397. [Abstract] [Full Text] [PDF]
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G. Sun, M. A. Stacey, M. Schmidt, L. Mori, and S. Mattoli Interaction of Mite Allergens Der P3 and Der P9 with Protease-Activated Receptor-2 Expressed by Lung Epithelial Cells J. Immunol., July 15, 2001; 167(2): 1014 - 1021. [Abstract] [Full Text] [PDF]
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J. Reibman, A. T. Talbot, Y. Hsu, G. Ou, J. Jover, D. Nilsen, and M. H. Pillinger Regulation of Expression of Granulocyte-Macrophage Colony-Stimulating Factor in Human Bronchial Epithelial Cells: Roles of Protein Kinase C and Mitogen-Activated Protein Kinases J. Immunol., August 1, 2000; 165(3): 1618 - 1625. [Abstract] [Full Text] [PDF]
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N. A. Lee and J. J. Lee The Macroimportance of the Pulmonary Immune Microenvironment Am. J. Respir. Cell Mol. Biol., September 1, 1999; 21(3): 298 - 302. [Full Text]
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D. C. Tsitoura, R. H. DeKruyff, J. R. Lamb, and D. T. Umetsu Intranasal Exposure to Protein Antigen Induces Immunological Tolerance Mediated by Functionally Disabled CD4+ T Cells J. Immunol., September 1, 1999; 163(5): 2592 - 2600. [Abstract] [Full Text] [PDF]
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