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Departments of
*
Medicine and
Mechanical Engineering, University of Alberta, Edmonton, Alberta, Canada;
University of Pennsylvania School of Medicine, Philadelphia, PA 19104;
§
Department of Pediatrics, Chonbuk National University, Chonju, Chonbuk, Korea; and
¶
Institute for Medical Sciences, Dongsan Medical Centre, Taegu, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-receptor cross-linking induces Syk
PTK phosphorylation and activation, resulting in Syk-dependent events
required for phagocytosis and mediator release. We hypothesized that
Syk antisense oligodeoxynucleotides (ASO) delivered by aerosol to rat
lungs in vivo would depress Syk PTK expression, mediator release from
alveolar macrophages, and Syk-dependent pulmonary inflammation. RT-PCR
and RT-in situ PCR demonstrated that aerosolized Syk ASO administration
reduced Syk mRNA expression from alveolar macrophages compared with
cells isolated from sham-treated rats. Western blot analysis confirmed
that Syk PTK expression was reduced after Syk ASO treatment. Compared
with sham-treated rats (scrambled oligodeoxynucleotide), Syk ASO
treatment suppressed Fc-receptor-mediated nitric oxide (86.0 ±
8.3%) and TNF (73.1 ± 3.1%) production by alveolar macrophages
stimulated with IgG-anti-IgG complexes. In contrast,
Fc-receptor-induced IL-1ß release was unaffected by Syk ASO
treatment. Additionally, Syk ASO suppressed Ag-induced pulmonary
inflammation, suggesting that Syk ASO may prove useful as an
anti-inflammatory therapy in disorders such as
asthma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R leads to downstream
signaling events, gene transcription, mediator release, and
phagocytosis. In macrophages, cross-linking of FcR also results in
the activation of Src and Syk protein tyrosine kinases
(PTK).3 These protein tyrosine kinases associate
with specific recognition sequences, immunoreceptor tyrosine-based
activation motifs, present in the intracellular domains of FcR
(1, 2, 3, 4, 5).
In addition to being expressed in macrophages (6, 7), Syk
(p72Syk) PTK is expressed in eosinophils
(8), T cells (9, 10, 11), B cells (10, 11), neutrophils (12), and mast cells
(13, 14, 15). Recently, Matsuda et al. (16)
observed that in vitro treatment of human peripheral blood monocytes
with Syk antisense oligodeoxynucleotides (ASO) inhibited Syk mRNA and
protein expression compared with cells treated with scrambled
oligodeoxynucleotides (ODN). This inhibition correlated with the
suppression of FcR-mediated phagocytosis and indicated that Syk PTK
plays a critical role in FcR-mediated cellular signaling and
function in monocytes and macrophages. These data suggest that Syk ASO
may be effective in vivo in inhibiting Syk PTK expression and cellular
function in several cell populations including eosinophils, mast cells,
and macrophages.
Alveolar macrophages are the most abundant cell type in the airways and are important in host defense and the inflammatory response. Alveolar macrophages rapidly internalize liposomes and are likely to be exposed to Syk ASO after aerosolization (17).
Therefore, we studied the effects of Syk ASO liposome complexes delivered in vivo by aerosolization on a rat model of pulmonary inflammation. In these studies we also examined the effects of in vivo aerosolized Syk ASO liposome complexes on the expression of Syk PTK and on the function of alveolar macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Syk ASO and the scrambled ODN, respectively, were prepared by
the Nucleic Acid Facility of the Department of Chemistry at the
University of Pennsylvania Cancer Center and the Core DNA Services at
University of Calgary. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP)
and dioleoylphosphatidyl-ethanol-amine (DOPE) phospholipids were
purchased from Avanti Polar Lipids (Alabaster, AL). DOTAP liposomal
transfection reagent for in vitro studies was purchased from Boehringer
Mannheim (Indianapolis, IN). RPMI 1640, Eagle’s MEM (EMEM), FBS,
penicillin, and streptomycin were purchased from Life Technologies
(Grand Island, NY). Rat IgG2a (
) and mouse monoclonal anti-rat
IgG2a (used to form IgG-anti-IgG complexes) were purchased from
Serotec (Toronto, Ontario, Canada). Rabbit anti-murine Syk
polyclonal Ab was purchased from Upstate Biotechnology (Lake Placid,
NY), and the isotype control, polyclonal rabbit IgG, was purchased from
Serotec. F(ab')2 goat anti-rabbit HRP was
supplied by The Jackson Laboratory (Bar Harbor, ME). MTT, PMSF,
aprotinin, p-tosyl arginine methyl ester, and leupeptin were supplied
by Sigma (St. Louis, MO). Rat recombinant TNF and rat IL-1ß ELISA
kits were purchased from R&D Systems (Minneapolis, MN).
Chemiluminescence reagent was purchased from DuPont NEN (Boston,
MA).
Animals
Male Sprague Dawley rats were used throughout this study. They were housed in the University of Alberta animal housing facility in filter-top cages to minimize unwanted infection. They were exposed to 12-h light/dark cycles and were given food and water ad libitum. All rats were infected by s.c. injection of 0.5 ml PBS containing 3000 larvae (L3 stage) of Nippostrongylus brasiliensis and were studied 4 wk later (18). The worms are expelled by the host within 2–3 wk, and the inflammation in the lungs and the intestine subsides, leaving rats sensitized for re-exposure experiments. This work was approved by the University of Alberta Animal Ethics Committee in accordance with guidelines of the Canadian Council for Animal Care.
Syk ASO and scrambled ODN
A stem-loop rat Syk ASO was designed as described by Matsuda et al. (16) for human Syk ASO. The 60 base ASO consisted of the following sequence: 5'-GCCGCGGTTGCCCGCCATGTCTGATTTGATTCTTGAGATTTGGTAGTATCCCTCCGCGGC-3'. The scrambled ODN, also a 60-basesequence, has the same content of A, T, G, and C, and consisted of the following sequence: 5'-GCCGCGGTTCCTGAGTGTATCTGATTCGTACGATTCTATGGTCGTCGTCTCATCCGCGGC-3'. The stem-loop ODN and the scrambled ODN have three phosphorothioate backbone modifications, which increases their resistance to nuclease degradation. The stem-loop ASO is designed to interact with Syk mRNA at three sites, increasing its efficiency (16).
Liposome preparation
Liposomes were prepared using a method modified from Legendre
and Szoka (19). One milligram DOTAP in chloroform and 1 mg
DOPE in chloroform were mixed, and the chloroform was evaporated,
leaving a film of 1:1 DOTAP:DOPE. Two milliliters of sterile saline
were added, resulting in liposome formation composed of equal parts of
DOTAP and DOPE. The liposomes varied in size and were dispersed in an
ultrasonic bath (for 
1 min) until they were 200 nm in diameter.
The diameters of the liposomes suspended in saline were determined by
dynamic light scattering using a Brookhaven (BI90) particle sizer
(Brookhaven Instruments, Holtsville, NY).
Incubation of liposomes with Syk ASO
Because DOTAP:DOPE liposomes are cationic, they form complexes with negatively charged ODN (20). Complexes were formed by incubating a 2.5:1 ratio of the liposomes (1.25 mg) with either Syk ASO (0.5 mg) or the scrambled ODN (0.5 mg) for 45 min at room temperature in sterile saline (final volume, 9 ml). The ratio of liposome:Syk ASO was optimized as described below.
In vitro studies of Syk ASO
To examine rat Syk ASO in vitro, we studied the effects on Syk expression in the rat basophilic cell line RBL-2H3. RBL-2H3 cells were grown in EMEM supplemented with 17% FBS, 100 U penicillin, and 100 µg/ml streptomycin, and 4 mM glutamine at 37°C in 5% CO2. A total of 1 x 106 RBL-2H3 cells were seeded in each well of a 24-well plate. Twenty-four hours later, ASO/liposome complexes were added using complexes formed between Syk ASO and DOTAP transfection agent. In vitro, RBL-2H3 cells readily take up Syk ASO when it is complexed with DOTAP liposomes. With RBL-2H3 cells in vitro, there is no need to include in the liposome formulation the lipid DOPE, which was used in vivo to enhance Syk ASO delivery. Each addition consisted of 2 µl DOTAP (1 mg/ml stock) and 1 µl Syk ASO (1 mg/ml stock), which were allowed to form complexes in EMEM (75 µl total volume). The ASO-liposome complexes (75 µl) were then added to each well containing 175 µl culture medium without serum and the cells were incubated at 37°C for 24 h. A second volume of ASO-liposome complexes (75 µl) was then added, the culture medium was adjusted to 5% FBS (final volume, 1.0 ml), and the RBL-2H3 cells were incubated for 1 day before study. After treatment with liposome, scrambled ODN-liposome complexes, or Syk ASO-liposome complexes, cells were used for RT-PCR analysis of Syk mRNA expression and Western blot analysis of Syk protein expression.
After control and ASO-liposome treatment, a histamine release assay was
also performed by cross-linking the RBL-2H3 IgE receptor Fc
RI using
J17 (murine anti-rat FcRI Ab) and measuring the resulting
histamine release by enzyme immunoassay kit (Immunotech, Marseille,
France) as described below. Twenty-four-well plates containing 1
x 105 RBL-2H3 per well in 1.0 ml EMEM were
incubated overnight at 37°C. The cells were washed once with PBS and
incubated on ice with 1.0 ml PAGCM (histamine release buffer) and 100
µl of the anti-rat FcRI Ab for 30 min at 4°C. After one wash
with PBS, the RBL-2H3 cells were incubated as follows: 1.0 ml PAGCM
alone (negative control), 1.0 ml PAGCM containing 10 µl calcium
ionophore (50 µg/ml stock) (positive control), or 1.0 ml PAGCM
containing 10 µl murine anti-rat FcRI Ab (1 mg/ml) for 30 min
at 37°C. The PAGCM containing histamine was removed from the cells
and assayed by enzyme immunoassay. Standards were included to establish
a dose curve for histamine.
Aerosolized administration of Syk ASO and harvesting of alveolar macrophages
To restrict their movement, animals were confined in plexiglass
animal chambers, which were then placed in small plastic boxes with
lids for the duration of aerosolization. Nine milliliters of liposome,
scrambled ODN/liposome, or Syk ASO/liposome complexes were administered
by nebulization for 30 min using a Sidestream nebulizer, model 1200A
durable (Medic-Aid, Pagham, U.K.). Twenty-four hours later the
procedure was repeated, and another 24 h later the animals were
anaesthetized with an i.p. injection of 0.5 ml Rompun (20 mg/ml) and
0.5 ml Ketalean (Ketamine, 100 mg/ml) and then sacrificed by severing
of the abdominal aorta.
The trachea of each rat was cannulated with polyethylene tubing
(1.14-mm diameter; Becton Dickinson, Sparks, MD) attached to a needle,
and 5 ml of ice-cold PBS was massaged into the lungs 12 times as
described previously (21). The PBS was aspirated into
ice-cold polypropylene tubes. The cells were kept on ice until they
were washed by centrifugation at 150 x g for 20 min
and resuspended in PBS, yielding 
95% alveolar macrophages as
determined by staining of cytospins with May-Grünwald-Geimsa
stain. Cell viability was >95% as determined by trypan blue
exclusion. The isolated macrophages were then studied 1) for RNA
isolation and RT-PCR analysis of Syk mRNA expression, 2) by Western
blot analysis of Syk protein expression, or 3) by analysis of mediator
release.
Optimization of aerosol Syk ASO delivery
To confirm that the aerosolization procedure delivered particles to the lower airways, methylene blue was aerosolized. On frozen tissue sections of lung, areas in and around the bronchioles were stained blue, confirming that the nebulizer delivered dye to the lower airways. In addition, the nebulizer produced methylene blue droplets of 5.1 ± 0.4 µm, which are small enough to enter the lower airways, further validating the aerosolization system. Nebulized Syk ASO/liposome complexes had droplet sizes of 4.1 ± 0.1 µm, similar to those of methylene blue, suggesting that ODN/liposome droplets were small enough to penetrate the lower airways. Particle sizes were measured using Doppler anemometry (Dantec Electronics, Mahwah, NJ) at the exit of the nebulizer.
Optimization of liposome/Syk ASO ratio and concentration
Because i.p. delivery controls the precise dose of Syk ASO administered and because this may not be the case with aerosolization, we used data from i.p. treated rats to optimize liposome:Syk ASO ratio and concentration. We examined the ability of varying liposome:Syk ASO ratios and concentrations to inhibit IgE-mediated (Syk-dependent) histamine release from purified peritoneal mast cells (22). The optimum ratio and Syk ASO concentration was identified as having the strongest inhibition of IgE-mediated histamine release. Liposome:Syk ASO (2.5:1) delivered i.p. (results not shown) was found to be the optimum ratio, as reported earlier by Zelphati and Szoka (23), for studies of maximal cellular uptake of antisense in vitro. In a similar manner, the optimum concentration of Syk ASO administered i.p. was determined to be 0.25 mg ASO/animal/day.
However, because a large proportion of aerosolized Syk ASO is not inhaled by the rats in our aerosolization delivery system, we increased the aerosol dose of Syk ASO or scrambled ODN to 0.5 mg/animal/day for all experiments using aerosol delivery and maintained the 2.5:1 liposome:Syk ASO ratio.
RNA isolation and solution-phase RT-PCR
Total RNA was isolated from alveolar macrophages and RBL-2H3 using TRIzol reagent (Life Technologies, Burlington, Canada) and quantified using an OD260/280 ratio, frozen at -70°C until RT-PCR analysis of Syk RNA expression. One rat per treatment group provided sufficient alveolar macrophages to yield at least 1 µg total RNA. RNA was reverse transcribed by SuperScript RNase (Life Technologies, Grand Island, NY) using a PTC-100 programmable thermal controller (MJ Research, Cambridge, MA) according to the manufacturer’s protocols. PCR was modified from the Life Technologies, (Burlington, Canada) Taq DNA polymerase protocol, with changes in the concentration of dNTP (1.23 mM) and 10x PCR buffer (67 mM Tris (pH 8.8), 1.5 mM MgCl2, 16.6 mM (NH4)2SO4, and 10 mM BME) in a total volume of 20 µl. Semiquantitative PCR was initially performed using increasing mRNA and cDNA concentrations to determine the appropriate optimal PCR cycle numbers. After preliminary study of PCR cycle numbers, 27 cycles were used (95°C for 45 s, 62°C for 45 s, and 72°C for 2 min). Twenty-seven PCR cylcles were performed with synthesized cDNA as templates using two primers, Rat-5' Syk, 5'-TTTGGCAACATCACCCGG-3'; and Rat-3' Syk, 5'-ACTTATGATGGCTTGCTC-3'. Products were run on a 2% agarose gel and were stained with ethidium bromide, producing a 400-bp Syk product. Controls included histidine decarboxylase primers Rat-5' histidine decarboxylase (HDC), 5'-GGAGCCCAGTGAATACCATG-3'; and Rat-3' HDC, 5'-TGCAGAGGACTGTCAGCGAA-3' (460-bp HDC product), for in vivo-treated alveolar macrophages and in vitro-treated RBL-2H3.
RT-in situ PCR
RT-in situ PCR was modified and performed as previously described (24). The macrophages were fixed for 16 h in 10% buffered neutral formaldehyde (BDH, Toronto, Ontario, Canada) at 22°C, washed twice with diethyl pyrocarbonate-treated water, and then placed on silane-coated glass slides (Perkin-Elmer, Norwalk, CT), each with three spots for test, positive, and negative controls. The cells were allowed to dry overnight, were digested in 10,000 U/ml pepsin (Boehringer Mannheim, Mannheim, Germany) in 0.01 M HCl for 1 h at 22°C, and then were treated with RNase-free DNase I (2,000 U/ml; Boehringer Mannheim, Indianapolis, IN) at 37°C for 24 h. The test specimens were treated with a RT solution containing 1 µM antisense primer or 25 µg/ml oligo(dT)12–18 primer (Life Technologies, Burlington, Canada) with Moloney murine leukemia virus RT (Life Technologies, Burlington, Canada) for 3 h at 37°C. Amplification of the cDNA was accomplished using a hot start method (24) with a PCR solution containing 4.5 mM MgCl2; 80 µM each of dATP, dCTP, dGTP, and dTTP; 0.8 µM of each primer; 16 µM of digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN), and 120 U/ml Taq polymerase (Life Technologies, Burlington, Canada) using a GeneAmp In Situ PCR System 1000 (Perkin-Elmer). Cycling conditions were 5 min at 94°C and 30 cycles of 94°C for 1 min, 64°C (ß-actin), 51°C (Syk) for 1 min, and 72°C for 1.5 min. The digoxigenin-11-dUTP-labeled PCR product was detected after incubation with alkaline phosphatase anti-digoxigenin conjugate (Boehringer Mannheim, Indianapolis, IN) (3.75 U/ml in 0.1 M Tris-HCl and 0.15 M NaCl (pH 7.5)) for 30 min at 22°C and after development in 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim, Indianapolis, IN) substrate solution. Test spots with DNase digestion and the RT step showed target mRNA expression in the cytoplasm. Positive controls without DNase treatment to monitor the length of protease digestion showed nuclear DNA priming, and negative controls, in which the cells were treated with DNase and the RT step was eliminated, showed that no priming of genomic DNA was detectable. The primers used for RT-in situ PCR Syk detection were the same as for solution-phase RT-PCR as described in the previous section. ß-Actin controls were performed for RT-in situ PCR using the following primers: Rat ß-actin 5' primer, 5'-GTGGGGCGCCCCAGGCACCA-3'; and 3' primer; 5'-GTCCTTAATGTCACGCACGATTTC-3' (526-bp ß-actin product).
Western blot analysis of Syk protein in RBL-2H3 cells
Cultured RBL-2H3 cells were treated with liposome alone (2 µg), 2 µg of control scrambled ODN and liposome, or Syk ASO and liposome at 1 x 105 cells/ml as described above. Cell lysates were prepared from 5 x 106 cells by boiling in Laemmli sample buffer (2% SDS, 10% glycerol, 100 mM DTT, and 60 mM Tris (pH 6.8)) for 5 min. Protein was loaded from lysates of equal cell numbers, separated by SDS-PAGE (12.5% polyacrylamide gel), and transferred to a nitrocellulose membrane in sample buffer (25 mM Tris, 190 mM glycine, and 20% methanol). The membrane was then incubated overnight at 4°C with 1 µg/ml rabbit anti-murine Syk polyclonal Ab before incubation (1.5 h at room temperature) with goat anti-rabbit HRP. Bands on the membrane were visualized with chemiluminescence reagent according to the manufacturer’s protocol. After detection of Syk protein, the Abs were removed by incubating the membrane in a stripping buffer containing 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCL (pH 6.7) for 30 min at 50°C with occasional agitation. The membrane was then reprobed with an anti-actin Ab (Actin I-19; Santa Cruz Biotechnology, Santa Cruz, CA). Bands on the membrane were visualized with chemiluminescence reagent.
Western blot analysis of Syk protein in alveolar macrophages
Alveolar macrophages from sham- and Syk ASO-treated animals were lysed for 1 h at 4°C with 1% Nonidet P-40 in the presence of the following protease inhibitors: 100 µg/ml PMSF, 5 µg/ml aprotinin, 5 µg/ml p-tosyl arginine methyl ester (serine protease inhibitors), and 5 µg/ml of leupeptin (thiol protease inhibitor). Protein extracted from 0.5 x 106 cells was separated on 10% SDS-PAGE and blotted onto Hybond-C Super membrane (Amersham, Oakville, Ontario, Canada). The membrane was blocked for 1.5 h in 3% skim milk and 5% goat serum at room temperature. The membrane was then incubated overnight at 4°C with 1 µg/ml rabbit anti-murine Syk polyclonal Ab before incubation (1.5 h at room temperature) with goat anti-rabbit HRP. Isotype controls were performed using purified polyclonal rabbit IgG as the primary Ab. Bands on the membrane were visualized with chemiluminescence reagent according to the manufacturer’s protocol.
Release and measurement of NO from alveolar macrophages
Rat alveolar macrophages (2 x 105 cells/well) suspended in complete RPMI 1640 medium (5% FBS) were allowed to rest for 1 h in a humidified incubator (37°C and 5% CO2) before stimulation for 24 h with IgG-anti-IgG complexes to induce NO production. IgG-anti-IgG complexes were prepared by incubating (37°C for 10 min) 100 µg/ml IgG2a with 20, 50, and 100 µg/ml mouse monoclonal anti-rat IgG2a. These complexes were resuspended and 20 µl was added to cells to make a final volume of 200 µl and final concentrations of 2, 5, and 10 µg/ml mouse anti-rat IgG2a. Because alveolar macrophage NO is rapidly converted to nitrite (NO2-), the Greiss method of NO2- detection was used to determine NO production. Cell-free supernatants were mixed with equal volumes of a 1:1 mixture of 1% sulfanilamide:0.1% N-(1-napthyl) ethylenediamine dihydrochloride dissolved in 2.5% H3PO4 and incubated for 10 min at room temperature (25). NO2- concentration was determined at 540 nm with a Molecular Devices Vmax kinetic microplate reader (Molecular Devices, Menlo Park, CA). NaNO2 (0.85–100 µM) was used to establish a standard curve for every experiment.
Release and measurement of TNF and IL-1ß from alveolar macrophages
Purified alveolar macrophages (2 x 105 cells/well) in complete RPMI 1640 medium were added to each well of a 96-well plate and were allowed to rest for 1 h in a humidified incubator (37°C and 5% CO2). The cells were then stimulated with IgG-anti-IgG complexes for 6 h and 24 h to induce TNF and IL-1ß production, respectively, as described for NO release experiments. The bioactivity of TNF was tested for cytotoxicity toward WEHI 164.13 using the MTT assay previously described (26). Briefly, 50 µl/well of rat recombinant TNF standard or samples were added into flat-bottom Linbro 96-well plates. Each sample was tested in duplicate. Eight two-fold serial dilutions starting from 100 pg/ml were used to establish the standard curve. Fifty microliters/well of 1 x 105 WEHI 164.13 cells/ml in RPMI medium supplemented with 10% FBS and 50 µM 2-ME was added and incubated for 20 h. Then, 10 µl/well of MTT (5 mg/ml) was added and further incubated for 3 h. Isopropanol-HCl (150 µl) was used to dissolve the purple formazan precipitates. The plate was read on a Vmax kinetic microplate reader (Molecular Devices) at 570 nm. Abs (IgG-anti-IgG) did not interfere with the TNF assay.
IL-1ß released from macrophages was measured in cell-free supernatants using ELISA instructions provided by R&D Systems.
Pulmonary inflammation
A rat model of pulmonary inflammation was induced by i.v. injection via the tail vein of 100 µl of saline containing 50 worm equivalents of N. brasiliensis Ag per sensitized rat as described previously (21). Sham-challenged animals received 100 µl saline alone. Eight hours after Ag challenge, animals were sacrificed and cells from the bronchoalveolar spaces were harvested by lavage as described above. Total bronchoalveolar lavage (BAL) cell counts and cell differentials, expressed as a percentage of total cells, were obtained.
Statistical analysis
Data are expressed as mean ± SEM for n separate experiments. After ANOVA, statistical analysis was performed using a two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first determined the effects of Syk ASO in vitro using the cell
line RBL-2H3. Syk ASO substantially reduced the level of Syk mRNA in
RBL-2H3 compared with controls (Fig. 1
A). In contrast, the
expression of HDC mRNA was not affected by Syk ASO treatment (Fig. 1B).
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). In
addition, Syk ASO treatment of RBL-2H3 significantly reduced histamine
release from 1.9 ± 0.3 ng/105 cells to
0.5 ± 0.2 ng/105 cells (74% inhibition)
after FcRI cross-linking (n = 3; data not
shown).
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Treatment of rats in vivo on days 1 and 2 with Syk ASO/liposome
complexes inhibited alveolar macrophage Syk mRNA expression, as
determined by solution-phase RT-PCR, compared with sham treatment
(liposome alone or scrambled ODN/liposome complexes) (Fig. 3A). Syk ASO treatment had no
effect on control HDC mRNA expression (Fig. 3B).
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). Fig. 4 demonstrates
that the in situ Syk mRNA signal is detected in the cytoplasm of
alveolar macrophages (Fig. 4A1). In Fig. 4A1, the
nuclei were unstained, reflecting the removal of genomic DNA by DNase
digestion. In contrast, the absence of DNase digestion (Fig. 4A2) leads to nuclear staining in the positive control,
demonstrating that the PCR step is successful. In the negative control
(Fig. 4A3), when the cells were treated with DNase and the
RT step was eliminated, the cells were unstained, indicating that the
product detected in the cytoplasm of cells in the test spot (Fig. 4A1) was from cDNA and was not priming from contaminating
genomic DNA.
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B demonstrates the detection of in situ Syk and
ß-actin mRNA. Positive and negative controls were also performed as
described above (results not shown). Treatment of rats on days 1 and 2
with Syk ASO/liposome complexes suppressed alveolar macrophage Syk mRNA
expression (Fig. 4B3) compared with sham treatment (liposome
alone, Fig. 4B1,; or scrambled ODN/liposome complexes, Fig. 4B2). Similar staining intensity in Fig. 4, B4–B6, indicates that ß-actin mRNA expression
from alveolar macrophages after Syk ASO treatment (Fig. 4B6)
was not significantly different from that of macrophages isolated from
sham-treated animals (Fig. 4, B4 and B5).
In a similar manner, Western blot analysis confirmed that the same
2-day Syk ASO/liposome complex treatment inhibited Syk PTK expression
in macrophage cell lysates compared with cell lysates from sham-treated
animals (Fig. 5). Purified Syk protein,
supplied by the manufacturer of the rabbit anti-murine Syk Ab and
isolated from Jurkat cells, was used as a positive control for Western
blot analysis of Syk protein. Bands of 72 kDa were visible by Western
blot analysis of cell lysates from sham-treated animals. In contrast,
the 72-kDa Syk protein band was markedly reduced in cell lysates from
Syk ASO-treated animals. Isotype controls for the anti-Syk Ab
showed no detectable bands at 72 kDa (data not shown). Thus, RT-PCR and
Western blot analyses clearly indicate that Syk ASO treatment
suppresses Syk PTK mRNA and protein expression.
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R-mediated NO release
from alveolar macrophages
Alveolar macrophages were stimulated with IgG-anti-IgG
complexes containing 5 or 10 µg/ml anti-IgG2a and 10 µg/ml
IgG2a for 24 h, which resulted in the release of significant
levels of NO (measured as
[NO2-]). IgG2a in the absence
of anti-IgG2a (sham) failed to induce NO release above spontaneous
levels (Fig. 6). When rats were treated
with Syk ASO/liposome complexes (compared with control treatment with
scrambled ODN/liposome complexes) once daily for 2 days before alveolar
macrophage isolation and stimulation (10 µg/ml IgG-anti-IgG2a),
NO release was inhibited by 73.1 ± 3.1% when spontaneous NO
release was subtracted from FcR-mediated NO release (Fig. 6;
n = 4). There was no significant difference in
FcR-mediated NO release from alveolar macrophages after treatment
with scrambled ODN/liposome complex or liposome alone.
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R-mediated TNF and
IL-1ß release from alveolar macrophages
The effects of Syk ASO treatment on cytokine release from alveolar
macrophages were determined by measuring TNF and IL-1ß production
(Figs. 7 and
8). After liposome, scrambled
ODN/liposome complex, or Syk ASO/liposome complex treatment on days 1
and 2, alveolar macrophages were isolated by BAL and were stimulated
with IgG-anti-IgG2a complexes for 6 h (TNF) and 24 h
(IL-1ß).
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and 8;
sham) failed to induce TNF or IL-1ß release above spontaneous levels.
Syk ASO/liposome complex treatment compared with control treatment with
scrambled ODN/liposome complexes significantly inhibited
FcR-mediated (10 µg/ml IgG-anti-IgG2a) TNF release by
86.0 ± 8.3% when the spontaneous TNF release was subtracted from
the FcR-mediated TNF release (Fig. 7; n = 4–5).
There was no significant difference in FcR-mediated TNF release from
alveolar macrophages after treatment with scrambled ODN/liposome
complexes or liposome alone.
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;
n = 4). Effects of in vivo Syk ASO treatment on pulmonary inflammation
Using an established model of pulmonary inflammation, we studied
the effects of in vivo Syk ASO treatment on pulmonary inflammation as
measured by BAL cell number and differential. In these experiments,
rats were treated once daily for 2 days with Syk ASO/liposome
complexes, scrambled ODN/liposome complexes, or liposome alone before
i.v. saline (sham) or Ag challenge (Fig. 9; n = 5). In the
liposome treatment group, we observed a significant increase in the
total BAL cell number 8 h after Ag challenge (4.3 ± 0.3
x 106) compared with 8 h after sham
challenge (2.3 ± 0.4 x 106).
Similarly, the total BAL cell number from scrambled ODN/liposome
complex-treated rats 8 h after Ag challenge was increased
(4.9 ± 1.0 x 106) compared with
8 h after sham challenge (2.2 ± 0.3 x
106). By contrast, no significant increase in BAL
cell number was observed 8 h after Ag challenge (2.6 ±
0.4 x 106) compared with sham challenge
(2.0 ± 0.7 x 106) in the Syk
ASO/liposome complex treatment group.
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). Although Ag challenge induced an
increase in absolute numbers of lymphocytes, neutrophils, and
eosinophils, the relative abundance of each cell type did not change
significantly as the macrophage numbers increased proportionately at
8 h after Ag challenge (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ASO are anionic molecules that cross cell membranes poorly. Cationic
liposomes such as DOTAP therefore have been used to enhance delivery of
ASO to target cells (23, 29, 30). For example, the CFTR
gene has been complexed with DOTAP and successfully administered to and
expressed in the nasal epithelium of humans with cystic fibrosis
(30). Zelphati and Szoka (23) demonstrated
that optimal delivery occurs when the liposome contains an additional
lipid such as DOPE. Furthermore, when a cationic liposome composed of
1:1 DOPE:DOTAP is used, the ideal ratio of liposome:ASO was 2.5:1. Our
studies confirm this observation and use DOTAP-DOPE liposomes complexed
with Syk ASO at a ratio of 2.5:1 for in vivo studies. For in vitro
studies we used liposomes made of DOTAP alone complexed with Syk ASO at
a ratio of 2:1. We observed that in vitro delivery of Syk ASO to the
rat mast cell line RBL-2H3 suppressed Syk mRNA and protein expression
compared with control-treated cells. Furthermore, Syk ASO suppressed
the Syk-dependent FcRI-mediated histamine release from RBL-2H3
cells. This Syk ASO-mediated suppression of histamine release supports
the observation by Zhang et al. (31) that Syk tyrosine
kinase is required for optimal FcRI-dependent histamine release from
mast cells.
Next, we studied the use of Syk ASO in vivo by delivering it to rat
lungs by aerosolization. It is well-known that optimal FcR
stimulation of macrophages is Syk PTK-dependent (6, 16).
Similar to the in vitro effects on Syk PTK expression, we observed that
aerosolized Syk ASO suppressed alveolar macrophage Syk mRNA and protein
expression as well as immune complex-mediated NO and TNF release.
However FcR-mediated IL-1ß release from alveolar macrophages was
unaffected.
A previous report indicates that the administration of ASO directed at other targets in the lung may also be effective. Ag-induced bronchoconstriction in a rabbit model of asthma was inhibited after treatment with an ASO directed against the adenosine receptor (32). Our studies are the first to target the important intracellular signaling molecule Syk PTK in vivo.
It is of interest that there was no inhibition of FcR-mediated
IL-1ß release by alveolar macrophages after Syk ASO treatment. We
previously observed a similar differential regulation of TNF and
IL-1ß release after CD8
ligation of alveolar macrophages and
demonstrated that CD8-mediated IL-1ß release but not TNF release
was inhibited by the protein kinase C inhibitor Ro 31-8220
(33). Thus, different intracellular pathways may be
utilized for TNF and IL-1ß release, a result further suggested by our
observations with Syk ASO treatment. Alternatively, IL-1ß synthesis
and release may require lower levels of active Syk PTK than NO and TNF
release do.
Having observed that in vivo delivery of Syk ASO suppressed alveolar macrophage function, we studied the effects of Syk ASO in an in vivo model of systemic anaphylaxis and focused on pulmonary inflammation. It has been established that when rats previously sensitized to N. brasiliensis are challenged by i.v. injection of Ag isolated from this parasite, anaphylactic shock occurs, one aspect of which is pulmonary inflammation. One method for assessing pulmonary inflammation is to determine the Ag-mediated changes in BAL cell number and cell types found in the BAL. We observed that Syk ASO treatment suppressed the Ag-mediated (8 h) increase in BAL cell number, indicating that Syk ASO has anti-inflammatory properties in the lung. However, the proportions of different cell populations in BAL did not change 8 h after Ag challenge. In future studies, we will examine the effects of Syk antisense on changes in cell populations found in BAL at different times after Ag challenge. We will also determine whether aerosolized Syk ASO selectively inhibits pulmonary inflammation or if it affects other components of the systemic anaphylaxis.
These data extend the observations of Matsuda et al. (16) and pioneer the use of aerosolized Syk ASO in vivo to inhibit Syk mRNA and protein expression as well as the Syk-dependent activation of alveolar macrophages. Syk ASO may prove useful in the study of allergic disorders because cells such as eosinophils and mast cells express several Syk-dependent pathways of activation (8, 13, 14, 15). Thus, Syk ASO-mediated Syk suppression may prove useful in the development of therapy for inflammatory diseases of the airways, such as asthma and allergic disorders, in addition to being potentially beneficial at other sites of inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. Dean Befus, Astra Chair in Asthma Research, Glaxo-Heritage Asthma Research Laboratories, Department of Medicine, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada. E-mail address:
3 Abbreviations used in this paper: PTK, protein tyrosine kinase; ASO, antisense oligodeoxynucleotide; ODN, oligodeoxynucleotides; EMEM, Eagle’s MEM; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOPE, dioleoylphosphatidyl-ethanol-amine; HDC, histidine decarboxylase; BAL, bronchoalveolar lavage.
Received for publication April 27, 1999. Accepted for publication January 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. receptor cDNA induces T cells to become phagocytic. Proc. Natl. Acad. Sci. USA 91:10232.RI-mediated tyrosine phosphorylation and activation of 72 kDa protein tyrosine kinase, PTK72, in RBL-2H3 rat tumour mast cells. Proc. Natl. Acad. Sci. USA 89:9107. receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides. Mol. Biol. Cell 7:1095.[Abstract]
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