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Departments of
* Medicine,
Laboratory Medicine and Pathology, and
Pharmacy, University of Alberta, and
HistoBest Inc., Edmonton, Alberta, Canada; and
¶ University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R and Fc
R, leading to the release
of biologically active molecules in inflammation. We reported
previously that aerosolized Syk antisense oligodeoxynucleotides (ASO)
depresses Syk expression in inflammatory cells, the release of
mediators from alveolar macrophages, and pulmonary inflammation. To
study the effect of Syk ASO in allergic inflammation and airway
hyperresponsiveness, we used the Brown Norway rat model of OVA-induced
allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid
complex by aerosol to rats, significantly inhibited the Ag-induced
inflammatory cell infiltrate in the bronchoalveolar space, decreasing
both neutrophilia and eosinophilia. The number of eosinophils in the
lung parenchyma was also diminished. Syk ASO also depressed
up-regulation of the expression of 
2 integrins,
4 integrin, and ICAM-1 in bronchoalveolar lavage
leukocytes and reversed the Ag-induced decrease in CD62L expression on
neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar
lavage following Ag challenge was significantly inhibited. Syk ASO also
suppressed Ag-mediated contraction of the trachea in a complementary
model. Thus, aerosolized Syk ASO suppresses many of the central
components of allergic asthma and inflammation and may provide a new
therapeutic approach. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R and FcR leads to downstream signaling events, gene
transcription, mediator release, and in some cases phagocytosis. In
leukocytes, cross-linking of FcR results in the activation of Src and
Syk protein tyrosine kinases. These kinases associate with
immunoreceptor tyrosine-based activation motifs, which serve as
specific recognition sequences in the intracellular domain of FcR
(1, 2, 3). Syk plays an essential role in activation of
immune cells and lymphocyte development. In mast cells, this molecule
is involved in regulation of multiple intracellular signaling pathways,
leading to release of allergic mediators. Important downstream targets
of Syk include phospholipase C1, the activation of which results in
Ca2+ mobilization and eventually in NF-AT
activation. Syk also induces activation of the mitogen-activated
protein kinase cascade and generation of phosphatidylinositide
3-phosphate, which in turn regulate transcription factors necessary for
cytokine gene expression (4). In addition to being
expressed in macrophages (5, 6) and mast cells
(7, 8, 9), Syk (p72Syk) is expressed
in eosinophils (10), neutrophils (11), T
cells (12, 13), and B cells (13, 14).
Recently, it has been shown that Syk is also expressed in
nonhemopoietic cells: human fibroblasts (15), breast
epithelium (16), and rat hepatocytes
(17).
Matsuda et al. (18) demonstrated that treatment of
peripheral blood monocytes with Syk antisense oligodeoxynucleotides
(ASO)4 inhibits Syk
expression when compared with cells treated with scrambled ASO (Scr
ASO). This inhibition correlates with the suppression of
FcR-mediated phagocytosis, suggesting that Syk plays a critical role
in FcR-mediated cellular signaling and function in
monocytes/macrophages.
We have recently demonstrated that aerosolized Syk ASO in vivo suppresses Syk expression, the release of NO and TNF from macrophages, and pulmonary inflammation in an infection model of airway inflammation (19). In the current studies we used this short-term gene therapy approach to treat allergic inflammation in the airways in a Brown Norway rat model of OVA-induced asthma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Brown Norway rats (Harlan Sprague Dawley, Indianapolis, IN) and male Sprague Dawley rats (Charles River Breeding Laboratories, St.-Constant, Quebec, Canada) were housed in the Health Sciences Laboratory Animal Service (University of Alberta, Edmonton, Alberta, Canada) in filter-top cages. The animals were rested for a minimum of 1 wk before experimentation. They were exposed to 12-h light/dark cycles and given food and water ad libitum.
The Brown Norway rats were sensitized to OVA i.p. as described previously (20) and used on day 21 following sensitization. The Sprague Dawley rats were infected by s.c. injection of larvae of Nippostrongylus brasiliensis (Nb) and were studied 4 wk later (21) when the inflammation in the lungs had subsided, leaving rats sensitized for re-exposure experiments. This work was approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee in accordance with guidelines of the Canadian Council for Animal Care.
Preparation of ASO/liposome complexes
A stem loop rat Syk ASO was designed to interact with Syk mRNA at three sites, increasing its efficiency, as described by Matsuda et al. (18). The 60-base phosphorothioate ASO (Nucleic Acid Facility, Department of Chemistry, University of Pennsylvania, Philadelphia, PA) and the Scr ASO (Core DNA Services, University of Calgary, Calgary, Canada) were described elsewhere (19).
1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleoyl-phosphatidyl-ethanol-amine (DOPE) liposomes were prepared using a method modified from Legendre and Szoka (22), described in detail in Ref. 19 . Cationic DOTAP:DOPE liposomes formed complexes with negatively charged oligodeoxynucleotides (23) by incubating a 2.5:1 ratio of the liposomes (1.25 mg) with either Syk ASO (0.5 mg) or the Scr ASO (0.5 mg) in a final volume of 18 ml. The liposome:Syk ASO ratio was optimized as described by Stenton et al. (19).
Aerosolized administration of Syk ASO
We used exactly the same protocol, previously shown to inhibit both Syk mRNA and protein expression in rat alveolar macrophages (19). Animals were placed in plastic boxes with lids for aerosolization. Nine milliliters of saline, liposome, Scr ASO/liposome, or Syk ASO/liposome complexes were administered per rat by nebulization for 45 min using a Sidestream nebulizer (model 1200A durable; Medic-Aid, Pagham, U.K.). Twenty-four hours later the procedure was repeated, followed by a third treatment at 48 h (unless otherwise stated). Immediately after the third treatment, animals were challenged with aerosolized saline or 5% OVA in saline for 5 min. Twenty-four hours after challenge, the animals were sacrificed.
Bronchoalveolar lavage
The trachea of each rat was cannulated with polyethylene tubing attached to an 18-gauge needle, and 5 ml of ice-cold PBS was massaged into the lungs 12 times as described previously (24). The PBS was aspirated into ice-cold polypropylene tubes. The first 2 ml of bronchoalveolar lavage (BAL) fluid obtained was used to measure the TNF level. The cells were kept on ice until they were washed by centrifugation at 150 x g for 20 min and resuspended in PBS, yielding a cell viability of >95% as determined by trypan blue exclusion. The isolated BAL cells were counted and cell smears were prepared using a Cytospin (Thermo Shandon, Pittsburgh, PA). In several experiments cells were also used for flow cytometry. Cell differentials were determined by counting cytospins of the BAL cells following staining with HEMA 3 reagent (Biochemical Sciences, Swedesboro, NJ).
Lung histology
For histopathological analyses, rats were treated as above except that BAL was not performed. Instead, the lungs were inflated by instilling 5 ml of 10% buffered formalin (Fisher Scientific, Nepean, Ontario, Canada) via the tracheotomy. The trachea was tied closed and the inflated lung was carefully removed to avoid puncturing and placed in 10% formalin for 24 h. Sample block preparation, staining, and analysis were performed by the University of Alberta Hospital’s Department of Laboratory Medicine and Pathology. Briefly, the entire lung was processed, paraffin-embedded, sectioned at 4 µm, and stained with H&E.
The whole lung sections were then assigned a score of 0–4 (0, no inflammation; 1, mild inflammation; 2, moderate inflammation; 3, severe inflammation; 4, extreme inflammation), according to the inflammation present, by a pathologist who was blind to the various treatments. The mean inflammatory scores based on the presence of hemorrhage, congestion, edema (alveolar and interstitial), and inflammation (airway lumen, airway wall, alveolar, interstitial, and perivascular) were recorded.
For counting lung eosinophils, after performing BAL the left lung was removed and fixed immediately in 10% buffered formalin. All specimens were coded, processed, and analyzed in a standardized fashion by HistoBest (Edmonton, Alberta, Canada). Multispecimen tissue blocks were prepared as previously described (25). Sections (4-µm thick) were stained by the method of Luna (26) for the specific identification of eosinophils. Cell counting was performed at a x400 magnification in alveolar sites and throughout parenchyma, but not in microgranuloma areas, trying to avoid unrepresentative areas for the whole tissue. The number of eosinophils per cubic millimeter (n) was obtained by the following formula: n = (number of eosinophils counted per 1-mm2 area of tissue examined x 1000 µm)/(2 x average size of eosinophils plus section thickness (in micrometers)), as previously described (27). The average diameter of eosinophils was 10 µm (range, 8–12 µm) and the average field area examined was 0.10 mm2 (i.e., corresponding to the average volume of 0.95 ± 0.55 mm3).
Immunofluorescent staining and flow cytometric analysis
BAL cells were washed with ice-cold PBS and fixed with 5%
formalin for 5 min at room temperature. After fixation cells were
incubated overnight in PBS containing 5% skim milk and 0.1% BSA on
ice (blocking solution). The cell suspension (1 x
106 cells/ml) was then preincubated with rat Fc
block (BD PharMingen, Mississauga, Ontario, Canada) diluted 1/200 for
15 min and then with mAbs directly conjugated to FITC or PE for 1
h on ice. The following mAbs were used: CD11a (WT.1), CD11b/c (OX-42),
CD18 (WT.3), CD49d (Mr4-1), CD54 (1A29), CD62L (HRL1), OX-41, and
OX-52 Abs (all FITC conjugated); CD3 (G4.18), CD8 (OX-8), and
anti-granulocyte (RP-1) (all PE conjugated) Abs; and the
corresponding isotype-matched controls. All Abs except OX-41 and OX-52
were purchased from BD PharMingen; OX-41 and OX-52 were from
Serotec (Toronto, Ontario, Canada). After two washings in PBS
the cells were analyzed by flow cytometry using a FACSort (BD
Biosciences Mountain View, CA) with CellQuest software (BD
Biosciences). Individual populations of cells were gated according to
their forward and side light scatter characteristics and immunostaining
for cell type-specific markers (OX-41 for alveolar macrophages, rat
granulocyte marker (RP-1), and CD3 for T lymphocytes). Discrimination
of eosinophils from other cells in BAL was based on their high
autofluorescence in combination with small size (low forward light
scatter), high granularity (high side light scatter), and the lack of
expression of other cell type-specific markers.
ELISA of TNF
TNF levels were measured in the supernatant of the first 2 ml of
BAL. The procedure was conducted according to the manufacturer’s
instructions using a commercial ELISA kit (rat TNF-; BioSource
International, Camarillo, CA). The minimal detectable level of TNF by
this assay was <4 pg/ml.
Ag-induced tracheal contraction
We first used OVA-sensitized Brown Norway rats and challenged their isolated tracheas by perfusing OVA through the lumen of the trachea. However, OVA did not induce strong, reproducible contractions of tracheal smooth muscle. Therefore, we used a different sensitization and challenge model: Sprague Dawley rats sensitized to the helminth Nb and challenged with LPS-free Ag isolated from Nb (24). One or 10 worm equivalents (WE)/ml were used to challenge tissue.
Four weeks following sensitization to Nb, Sprague Dawley rats were treated with or without aerosol of Syk ASO/liposome complexes (250 µg Syk ASO per rat) as described above on day 1 and 24 h later on day 2 (250 µg Syk ASO per rat). On day 3 (48 h after the first Syk ASO treatment) rats were sacrificed. A segment of trachea, carefully dissected and cleaned of connective tissue (12 rings of cartilage), was mounted in a Krebs filled organ bath as described by Pavlovic et al. (28). After stabilization (1 h), the tracheas were contracted by luminal perfusion of increasing doses of acetylcholine (-log M: 8 to 2.5), resulting in a dose-response curve that indicated the concentration of acetylcholine required to produce maximal contraction. Using washout phases (1 h), the tissues were allowed to recover before being luminally perfused with Nb Ag, resulting in tracheal contraction (circular muscle of trachea). Contraction was quantified using a pressure transducer and the data were analyzed on a computer. To standardize the Ag-induced contraction among the animals, Ag challenge data were calculated and plotted as a percentage of the maximal acetylcholine contraction.
Statistical analysis
Data are expressed as mean ± SEM for n separate
experiments. For all studies, statistical analysis was performed using
a Mann-Whitney test (p 
0.05 was considered
significant).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We studied the effects of in vivo Syk ASO treatment on OVA-induced
pulmonary inflammation as measured by BAL cell number and differential.
OVA challenge induced a significant increase in the total cell numbers
in BAL compared with sham-challengedanimals (10.1 ±
2.4 x 106 vs 3.3 ± 0.6 x
106, p < 0.05) (Fig. 1
A). The same large
differences in cell numbers between OVA-challenged and sham-challenged
animals were observed in rats treated with aerosolized liposome
(10 ± 2.2 x 106 vs 2.8 ±
0.4 x 106, p < 0.05) and
Scr ASO/liposome complex (10.2 ± 1.3 x
106 vs 3.5 ± 0.6 x
106, p < 0.05). By contrast, no
significant increase in total BAL cell number was observed 24 h
post-Ag challenge in rats treated with Syk ASO/liposome complex
(4.8 ± 0.6 x 106 vs 3 ±
0.4 x 106, p > 0.05).
Thus, compared with Scr ASO treatment, Syk ASO inhibited OVA-induced
increase in total BAL cell number by 71%.
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B)
of macrophages, neutrophils, eosinophils, and lymphocytes in BAL.
Remarkably, following OVA challenge, the proportion of neutrophils and
eosinophils in BAL from Scr ASO-treated rats reached 23.7 ± 4.8
and 20.2 ± 4.7%, respectively, compared with 0 ± 0 and
0.1 ± 0.1% in a sham-challenged group (data not shown). Syk ASO
treatment significantly inhibited neutrophilia by 74.1% and
eosinophilia by 85.2% compared with BAL cells from Scr ASO-treated
animals (p < 0.05). Syk ASO treatment restored
the percentage of macrophages in the BAL of OVA-challenged rats to
81.4 ± 5.9%, compared with 54.2 ± 7.7% in BAL of
OVA-challenged Scr ASO-treated rats.
The absolute numbers of macrophages, neutrophils, eosinophils, and
lymphocytes in BAL of OVA-challenged rats treated with Syk ASO were
significantly lower than in Scr ASO animals challenged with the Ag
(Fig. 1B).
Effects of frequency of treatment with Syk ASO
Three treatments of OVA-sensitized rats with Syk ASO over 48 h significantly inhibited the influx of inflammatory cells in BAL. To determine whether two treatments over 24 h were also sufficient to inhibit pulmonary inflammation, we conducted the same experiments as above, but rats were treated once daily on 2 consecutive days with Syk ASO/liposome complex or Scr ASO/liposome complex. As positive controls for the effects of Syk ASO we treated some rats with Syk ASO/liposome complex or Scr ASO/liposome complex once daily for 3 consecutive days before sham or OVA challenge.
In animals that received two treatments with Scr ASO, the total number of BAL cells increased from 2.8 ± 0.6 x 106 to 8.5 ± 2.5 x 106 following OVA challenge (data not shown). When rats received two treatments with Syk ASO, OVA challenge induced a similar increase in total BAL cell number (from 2.6 ± 0.9 x 106 to 10.3 ± 1.3 x 106), suggesting that two treatments with Syk ASO over 24 h were insufficient to suppress pulmonary inflammation. Positive controls confirmed that three treatments with Syk ASO significantly suppressed (52.5% inhibition) OVA-induced increase in total BAL cell number (4.8 ± 0.6 x 106). In agreement with these results, two treatments with Syk ASO over 24 h failed to inhibit neutrophilia and eosinophilia induced by Ag challenge (data not shown).
Duration of anti-inflammatory effect of Syk ASO treatment
We studied the duration of the inhibitory effect of in vivo
treatment with Syk ASO on OVA-induced pulmonary inflammation. Rats were
treated three times with Syk ASO over 48 h as described above, but
then were challenged with OVA immediately after the third treatment
(day 3 after the beginning of the treatment), 24 h later (day 4),
or 48 h later (day 5). BAL was performed and BAL samples were
obtained 24 h after the Ag challenge (i.e., on days 4, 5, and 6
after the beginning of the treatment). As shown on Fig. 2, significant reductions in total BAL
cell numbers, as well as in numbers of macrophages, lymphocytes,
neutrophils, and eosinophils as compared with the positive control,
were observed on days 4 and 5 after the Syk ASO treatment but not on
day 6. This suggests that the anti-inflammatory effect of Syk ASO
lasted for 2 days after three treatments over 48 h, but on day 3
it was gone.
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We found a 2.2-fold increase in TNF levels in BAL of
OVA-sensitized rats 24 h following Ag challenge (Fig. 3). In animals treated with Syk ASO and
challenged with the Ag immediately after the last treatment (day 3,
with BAL on day 4), TNF levels were significantly lower as compared
with rats treated with aerosolized saline. When the BAL was delayed
until the days 5 and 6 following the beginning of Syk ASO treatment (Ag
challenge on days 4 and 5), the treatment did not have a significant
effect on Ag-induced TNF production (Fig. 3).
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We studied the effects of Syk ASO on surface expression of several
adhesion molecules belonging to different families:
4 integrin (CD49d), 2
integrin (CD11a, CD11b/c, CD18), Ig superfamily (ICAM-1 or CD54), and
L-selectin (CD62L) in BAL cells obtained from OVA-sensitized rats
challenged with OVA or saline. As shown in Fig. 4, OVA challenge in sensitized animals
caused an increase in the percentage of BAL leukocytes expressing
CD49d, CD11a, CD11b/c, CD18, and CD54 on their surface as compared with
rats challenged with saline. The increase in CD49d, CD11a, and CD18 on
eosinophils and neutrophils as well as in CD11b/c and CD54 on
eosinophils, neutrophils, and macrophages reached statistical
significance. The percentage of neutrophils expressing CD62L decreased
following the Ag challenge, indicating the shedding of L-selectin upon
leukocyte activation.
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4 and 2 integrins on
eosinophils and neutrophils, as well as of ICAM-1 on neutrophils. In
macrophages, Syk ASO treatment significantly inhibited up-regulation of
the expression of both CD11b/c and CD54. The treatment also reversed
Ag-induced decrease in L-selectin expression on neutrophils (Fig. 4).
However, when animals were treated with OVA and Scr ASO/liposome
complex, or OVA and liposome alone, there was no effect on the cellular
expression of the adhesion molecules in BAL compared with treatment
with OVA alone (data not shown). Effects of Syk ASO on lung histopathology in OVA-challenged animals
We assessed whether Syk ASO treatment could suppress airway
inflammation in tissues by examining the histology of rat lungs (Fig. 5). Sensitization of rats to OVA in the
absence of subsequent Ag challenge did not affect the inflammatory
score 4 wk later (data not shown). Similarly, OVA-sensitized and
saline-challenged rats did not have significant signs of pulmonary
inflammation or had mild changes. Some control animals had rare
microgranulomas similar to that previously described in normal Brown
Norway rats (29). The mean inflammatory score
significantly increased from 1 ± 0.1 (sensitized and
saline-challenged rats) to 2.5 ± 0.1 when sensitized rats were
challenged with OVA (Fig. 5, A–C vs
D–F). In this case, numerous microgranulomas,
prominent inflammatory peribronchial and perivascular infiltration,
hemorrhage, congestion, and alveolar and interstitial edema were
observed. A similar magnitude of increase in inflammatory score was
observed in the lungs of OVA-challenged Scr ASO-treated rats (data not
shown). Moreover, using the scoring system devised, we could not detect
any statistical difference in pulmonary inflammation between Syk
ASO-treated and saline-treated animals challenged with OVA (Fig. 5, G–I).
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).
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We used OVA-sensitized Brown Norway rats and challenged their
isolated trachea by perfusing OVA through the lumen. However, a range
of concentrations of OVA (1–10 µg/ml) did not contract the isolated
trachea. Therefore, we applied a different sensitization and challenge
model. Sprague Dawley rats were sensitized to Nb and
challenged by luminal perfusion with Ag isolated from Nb as
described above. The maximum acetylcholine contraction of sensitized,
non-Syk ASO-treated and Syk ASO-treated rats was 2228 mg per gram of
tissue (n = 13) and 2109 mg per gram of tissue
(n = 4), respectively. Therefore, Syk ASO had no effect
on the acetylcholine-mediated contraction of the airway, a mechanism
believed not to involve Syk tyrosine kinase. As shown in Fig. 7, the sensitized isolated trachea
contracted consistently to Ag challenge with 1 and 10 WE/ml
(n = 6–8). Trachea from unsensitized animals did not
contract following Ag challenge, but contracted to acetylcholine
(maximum contraction: 2040 mg per gram of tissue), indicating
that Ag-mediated contraction occurred only after sensitization.
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). Thus,
Syk ASO treatment suppresses tracheal contraction in response to Ag
challenge. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R and FcR
(1, 2, 3). Syk induces tyrosine phosphorylation of multiple
intracellular proteins participating in signaling pathways of allergic
inflammation, such as Ca2+ mobilization and
mitogen-activated protein kinase cascade (2, 4). The
critical role of Syk in FcR-mediated signaling suggests that inhibition
of this molecule can affect allergic inflammatory responses. Indeed, we have recently observed that aerosolized Syk ASO-liposome complexes suppress alveolar macrophage Syk mRNA and protein expression, Syk-dependent TNF and NO release from alveolar macrophages, and pulmonary inflammation following i.v. Ag challenge in Nb-infected Sprague Dawley rats (19). Treatment of target cells with ASO requires an appropriate delivery system because ASO are anionic molecules that cross cell membranes poorly. We used cationic liposomes, DOTAP in combination with a neutral carrier lipid (DOPE), known to enhance delivery of ASO to target cells (30, 31). Our observations suggest that Syk may be a target for gene therapy in asthma and pulmonary inflammation.
To test this hypothesis, we applied the Brown Norway rat OVA-induced asthma model (32, 33, 34), using the same protocol previously shown to inhibit both Syk mRNA and protein expression (19). Aerosolized Syk ASO delivered in liposome complexes significantly inhibited pulmonary inflammation induced by Ag challenge in OVA-sensitized animals. Neither Scr ASO nor liposome alone caused this effect, eliminating the possibility of nonspecific mechanisms involved in the treatment.
Despite the inhibitory effect of Syk ASO on inflammatory cell infiltration in the bronchoalveolar space, treatment did not appear to affect major histopathological features of lung inflammation caused by Ag challenge. One possible explanation is that the scoring system used to evaluate the lung inflammation was not sensitive. More detailed histological analysis revealed that Syk ASO greatly inhibited eosinophil infiltration in lung parenchyma. Based on these observations we suggest that Syk ASO may inhibit eosinophil extravasation induced by Ag challenge. As a consequence we observed lower eosinophilia in BAL of Syk ASO-treated animals. This is significant because eosinophils are a major source of inflammatory mediators causing tissue damage and airway hyperresponsiveness in allergic asthma (35).
To complement the data on the effect of Syk ASO on airway smooth muscle responsiveness, we investigated Ag-mediated contraction of an isolated trachea from Sprague Dawley rats sensitized to Nb. Syk ASO treatment significantly suppressed Ag-mediated, but not acetylcholine-mediated, tracheal contraction. Acetylcholine contracted tracheal smooth muscle in a Syk-independent manner, whereas the effect of Nb Ag involved Syk-mediated signaling events.
Interestingly, in sensitized animals we could not induce tracheal contraction using OVA, although Ag isolated from Nb had a potent effect on tracheal smooth muscle. It appears that OVA challenge induces strong inflammatory responses in the lungs but does not directly affect tracheal smooth muscle. It is possible that inflammatory cells present in the lungs following Ag challenge release mediators potentially leading to smooth muscle contraction, but some down-regulating factors are also released and interfere with this effect. We suggest that there may be some negative regulators preventing the response of smooth muscle to the Ag challenge in this system, e.g., PGs. Thus, we have earlier demonstrated that the PG synthetase inhibitor indomethacin can suppress intestinal responsiveness to Nb Ag (36).
One hallmark of allergic asthma, namely inflammatory cell infiltration into the bronchoalveolar space, was used to evaluate the effect of different regimens of Syk ASO treatment. Three treatments with Syk ASO in a 48-h period, but not two treatments in a 24-h period, were sufficient to suppress pulmonary inflammation induced by Ag challenge. When three treatments with Syk ASO were provided, the anti-inflammatory effect of ASO lasted until day 5 after the beginning of the treatment, but on day 6 it was no longer evident. ASO appears to have a prolonged effect blocking adequate transcription and translation of Syk kinase for 48 h following its application, but after this time adequate transcription and translation are likely restored.
In another readout system, namely airway smooth muscle responsiveness in Nb-infected Sprague Dawley rats, two treatments with Syk ASO during 48 h appeared to be sufficient to suppress Ag-mediated contraction of the isolated trachea. These data agree with our previous observations that two treatments with Syk ASO during 48 h inhibited Nb-induced pulmonary inflammation (19). The model of pulmonary inflammation induced by Nb infection is apparently more sensitive to the inhibitory effect of Syk ASO compared with the Brown Norway rat OVA-induced asthma model. The effect of Syk ASO on tracheal smooth muscle contraction induced by Ag challenge can mostly be mediated by its anti-inflammatory action, but it is also possible that the antisense can have a direct effect on smooth muscle.
By what mechanisms does Syk ASO inhibit inflammatory cell infiltration into the bronchoalveolar space? Inflammatory cell infiltration into the airways is a complex process involving numerous interactions between cells and among cells and extracellular matrix proteins. These interactions, mediating leukocyte trafficking into the inflammatory site, involve several adhesion molecules (37, 38, 39, 40, 41, 42).
A crucial role of cellular adhesion molecules in accumulation of
leukocytes in the airways following antigenic challenge in OVA-induced
asthma in Brown Norway rats has been demonstrated using Abs
specifically neutralizing 1 integrin (VLA-4)
(41), 2 integrins (LFA-1, Mac-1)
(20, 40), and ICAM-1 (41). In our study we
observed a marked up-regulation by Ag challenge of
4 and 2 integrin and
ICAM-1 expression on leukocytes in BAL. This may account for the great
influx of inflammatory cells, especially eosinophils and
polymorphonuclear neutrophils (PMN), into bronchoalveolar
space.
The magnitude of increase in adhesion molecule expression in BAL cells depended on the cell type. In contrast to eosinophils and PMN, expression of CD11a and CD18 on the macrophage population was not significantly increased upon Ag challenge, perhaps due to the heterogeneity of the macrophage population in BAL. The latter consists of monocytes recently recruited from blood vessels and resident macrophages in the parenchyma and in the airway lumen. Interestingly, the expression of adhesion molecules is higher on PMN in BAL than on peripheral blood PMN obtained from the same animal (M. Ulanova, unpublished observations). Although we did not address this question in the present study, one might suggest that numerous cell-cell and cell-extracellular matrix interactions during the process of leukocyte migration up-regulate the expression of integrins and ICAM-1 on the cell surface.
In contrast to other adhesion molecules, L-selectin is shed upon leukocyte activation (43), and this may explain the lower percentage of CD62L+ neutrophils found in BAL of Ag-challenged rats compared with sham-challenged animals. In the present study we noted that Syk ASO treatment modulates the expression of adhesion molecules on BAL leukocytes, suggesting that regulation of adhesion molecules is an important component of the anti-inflammatory effect of Syk ASO. In particular, Syk ASO can affect eosinophil-dependent mechanisms in allergic asthma and inflammation by down-regulation of CD11a, CD18, and CD49d, mediating migration of eosinophils from blood vessels to the bronchoalveolar space. As a result, lower eosinophil numbers in both lung parenchyma and BAL are present in Syk ASO-treated animals compared with saline-treated animals.
The role of adhesion molecules in pulmonary inflammation is not limited
to their involvement in recruitment of leukocytes into the airways.
Interaction of adhesion molecules with their ligands leads to
activation of intracellular signaling pathways. In particular,
engagement of 1 integrins induced tyrosine
phosphorylation of Syk and activation of NF-
B that ultimately led to
the expression of proinflammatory cytokines, particularly TNF
(44). TNF is widely involved in regulation of leukocyte
adhesion both directly inducing expression of E-selectin, P-selectin,
ICAM-1, VCAM-1, and 2 integrins, and by
stimulating proinflammatory cytokine and chemokine release
(36). Accordingly, we investigated levels of TNF in BAL.
Our observations indicate that TNF production in BAL, induced by Ag
challenge, is inhibited by Syk ASO. TNF was shown to be produced
shortly after Ag challenge (45), and the amount of this
cytokine that we measured in BAL is likely the net result of its
accumulation and loss during the time between antigenic challenge and
the BAL procedure (24 h). Our previous experiments demonstrated that
Syk ASO treatment inhibits TNF production in alveolar macrophages,
indicating a direct effect of ASO on TNF mRNA transcription
(19). Data from the present study expand these
observations in an in vivo system and suggest that aerosolized delivery
of Syk ASO leads to the decrease of TNF mRNA in alveolar macrophages
and perhaps other cells such as mast cells, epithelial cells, and
fibroblasts. The resulting decrease in TNF production may interfere
with up-regulation of adhesion molecules following Ag challenge. In
contrast, Syk may act in other ways, including an effect on the
transcriptional regulation of adhesion molecule expression. Our data
suggest that, after Syk ASO treatment, integrin signaling might be
impaired. However, dependence of the integrin signaling on Syk is still
an open question. Recent observations indicated that
complement-mediated phagocytosis involving integrins CD11b/CD18 and
CD11c/CD18 as complement receptors was intact in
Syk-/- macrophages (2).
Another unresolved question concerns target cells for Syk ASO delivered to the airways. Alveolar macrophages express both Syk mRNA and protein (19) and may be the primary target of Syk ASO. It is interesting that breast epithelial cell lines express Syk (16), and it may be that this signaling molecule is also expressed by airway epithelial cells and is a target of Syk ASO in our model.
The present data extend our previous observations suggesting that Syk ASO may be a useful anti-inflammatory agent. Syk ASO suppresses Ag-induced tracheal contraction as well as pulmonary inflammation in a model of asthma. Modulation of the expression of adhesion molecules in BAL cells and down-regulation of production of TNF by aerosolized Syk ASO may represent important mechanisms of its anti-inflammatory action. These findings may provide a basis for a new strategy in the anti-inflammatory therapy of human asthma.
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Acknowledgments |
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Footnotes |
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2 G.R.S. and M.U. are joint first authors of this paper.
3 Address correspondence and reprint requests to Dr. A. Dean Befus, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. E-mail address: dean.befus{at}ualberta.ca
4 Abbreviations used in this paper: ASO, antisense oligodeoxynucleotide; Scr ASO, scrambled ASO; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOPE, dioleoyl-phosphatidyl-ethanol-amine; BAL, bronchoalveolar lavage; Nb, Nippostrongylus brasiliensis; PMN, polymorphonuclear neutrophil; WE, worm equivalent.
Received for publication January 17, 2002. Accepted for publication May 10, 2002.
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receptor signaling in macrophages and neutrophils. Mol. Cell. Biol. 18:4209. receptors on macrophages. J. Exp. Med. 186:1027.RIIIA activates the receptor-associated protein tyrosine kinase Syk and induces phosphorylation of multiple proteins, including p95Vav and p62/GAP-associated protein. J. Immunol. 152:5429.[Abstract]
subunit of Fc receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J. Biol. Chem. 269:3897.RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells. Proc. Natl. Acad. Sci. USA 89:9107. chain after receptor aggregation. J. Biol. Chem. 268:23318. receptor cDNA induces T cells to become phagocytic. Proc. Natl. Acad. Sci. USA 91:10232. receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides. Mol. Biol. Cell 7:1095.[Abstract]
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