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Introduction
The interaction between central nervous-immune pathways or neuroendocrine-immune
(NEI) networks in various disease states is affected by various
pathophysiologic status. In the pathogenesis of allergic diseases,
the abnormalities in immune function are mediated by the NEI network
through overproduction of allergic mediators[1]. Undoubtedly,
leukotrienes (LTs), the metabolites of 5-lipoxygenase (5-LO) pathways
are thought to play crucial roles in the inflammatory network. Up
to now, it is unclear how the metabolites of 5-LO pathways in the
central nervous system regulate inflammation in lung tissue of asthma
rats. From previous work, we found that the change of Th1/Th2 paradigm
(ratio of interferon (IFN)-g/interleukin (IL)-4 decreased and the
content of LTB4 in the cerebral cortex increased correspondent
to that in bronchoalveolar lavage fluid (BALF) or lung tissue in
inflammatory status of asthma rats[2,3] . These findings
indicate that there is an interactive response of LTs and proinflammatory
cytokines in the central nervous system, and the changes of these
proinflammatory mediators in the central nervous system may have
pathophysiologic effects in asthma rats. To explore these hypotheses
and get a clearer understanding of the central nervous system changes
of 5-LO pathway, Tumor necrosis factor (TNF)-a, IL-4, IFN-¦Ã, and
nitric oxide (NO) in asthma rats, we have further investigated the
correlative alterations of proinflammatory mediators and the expression
of 5-LO pathway enzyme in lung tissue and cerebral cortex.
Materials and methods
Animal and reagent Sprague-Dawley rats of
either sex weighing 180 g±20 g were purchased from the Laboratory
Animal Center of Medical School of Zhejiang University (Grade II,
Certificate No 220010014), and maintained on a regular 12-h
daylight cycle with water and food available ad libitum.
Dexamethasone sodium phosphate (DXM, Suzhou Sixth Pharmaceutical
Factory, Suzhou), ketotifen (Shanghai Pharmaceutical Factory, Shanghai),
egg albumin Grade V (Sigma, St louis, USA), heparin sodium (Xuzhou
Biochemical Pharmaceutical Factory, Xuzhou), IFN-¦Ã, TNF-¦Á, NO, and
IL-4 ELISA kit (Jinmei Biotech Co Ltd, Shenzhen, China) were commercially
available. TRIzol reagent, SuperScript II reverse transcriptase,
and Taq DNA polymerase were obtained from GIBCO BRL (Paisley,
Scotland, UK).
Sensitization, treatment, and challenge regimens The sensitization
procedure to induce IgE-mediated asthma response is as earlier described[3].
At d 14 after sensitization, the rats were placed in
a 45 cm×45 cm×15 cm plastic box and challenged by exposure
to an aerosol of ovalbumin (10 g/L in saline) which was generated
in a jet nebulizer (partical size 1-5 µm; BARI, MASTER, Germany)
for 20 min, and repeated daily for 6 d. Control rats were similarly
exposed to an aerosol of saline. One hour before antigen challenge,
the rats in the treatment groups were administered with DXM (0.5
mg/kg, ip) and ketotifen (5 mg/kg, ig), respectively. The rats in
the control and model groups were administered with saline (ip).
All the animals were studied on d 21 after the first sensitization.
BALF Bronchoalveolar lavage (BAL) was performed by flushing
the airways with saline 5 mL containing 1% bovine serum albumin
and 1 kU/L heparin sodium through the tracheal cannula three times.
BAL fluid (BALF) were pooled and centrifuged (Eppendorf Centrifuge
5804R, Germany) at 500×g for 10 min at 4 oC.
The supernatant was collected and stored at -70 oC for
assaying IFN-¦Ã, TNF-¦Á, NO, and IL-4 level.
Brain homogenates preparation Blood cells in systemic circulation
were removed by perfused with D-Hanks' liquid through ascending
aorta cannula. The left cerebral cortex was then cut into 1 mm3,
homogenated (DY89-I Homogenater, Linbo Xinzhi SCI-TETH research
Institute, Ningbo) in ice-cold Hanks' buffer (pH 7.5). Thereafter,
the homogenates were centrifuged at 3000×g at 4 oC
for 10 min. The supernatant was collected and stored at -70 oC
for assaying IFN-¦Ã, TNF-¦Á, NO, and IL-4 level.
Assay of IFN-¦Ã, TNF-¦Á, NO, and IL-4 The IFN-¦Ã, TNF-¦Á, NO,
and IL-4 levels in the BALF and cerebral cortex homogenates were
measured according to the ELISA kit description. All measurements
were carried out in duplicate.
Reverse transcription-polymerase chain reaction (RT-PCR) assay
Total RNA was isolated from the cerebral cortex and lung tissue.
Trizol 1 mL was added per 100 mg tissue and was homogenized; samples
were left at 18-21 oC for 3 min. Chloroform 0.2 mL was
added to the homogenizer and mixed. After 10 min at 18-21 oC,
the samples were centrifuged at 15 000× g in a microcentrifuge
at 4 oC for 15 min. The upper phase was transferred to
new tubes and the same volume isopropanol was added. RNA was allowed
to precipitate at 18-21 oC for 10 min and collected by
centrifugation at 15 000×g at 4 oC for 10
min. After washing precipitates in 75% ethanol and drying for 10
min, the RNA was dissolved in DEPC-treated water and quantified
by the measurement of ultraviolet absorption at 260 nm (Eppendorf
Biophotometer, Germany). All samples were kept at -70 oC
until use.
For the synthesis of the single-stranded cDNA (Eppendorf Mastercycler
gradient, Germany), total RNA (4 mg) from each sample was reverse-transcripted
using an antisense specific primer and 200 U of SuperScript II RT.
Sequences of the PCR primers for 5-LO, LTA4-H, and glyceraldehydes-3-phosphate
dehydrogenase (GAPDH) were derived from published sequences as follows:
5-LO: sense 5´ AGG CTG CAG TGA GAA GCA TC 3´, antisense
5´ GCC AGT GGT TCT TGA CTC TC 3´, designed to amplify
a fragment corresponding to nucleotides 181-770[4]; LTA4-H:
sense 5´CAG TCA CAG GAG GAT AAT 3´, antisense 5´
GGA GTG AGC CAC TGA AGG 3´, designed to amplify a fragment
corresponding to nucleotides 131-363[5]; GAPDH: sense
5´ ACC ACC ATG GAG AAG GCT GG 3´, antisense 5´ CTC
AGT GTA GCC CAG GAT GC 3´, designed to amplify a fragment corresponding
to nucleotides 372-899. All amplified segments extended over at
least 1 intron boundary of the genomic DNA. The reactants were cycled
at 94 oC 30 s, 59 oC 20 s, 72 oC
10 s for LTA4-H, and 94 oC 45 s, 58 oC
30 s, 72 oC 90 s for 5-LO and GAPDH. Reaction products
were separated by electrophoresis using 1.5% standard agarose gel.
Amplification of GAPDH was performed as a control. PCR products
were analyzed by electrophoresis (CLP, USA), visualized by ultraviolet
transillumination of ethidium bromide-stained gels, and the intensity
of each band was measured by the UVP Image Analyzer (UVP, Inc Upland,
CA). The expression of 5-LO and LTA4-H were normalized
to that of GAPDH.
5-LO Western immunoblotting The cerebral cortex and lung
tissue were dissected and homogenized in homogenizing buffer, containing
Tris-HCl 20 mmol/L, egtazic acid 2 mmol/L, edetic acid 5 mmol/L,
pepstatin 1.5 mmol/L, leupeptin 2 mmol/L, phenylmethylsulfonyl fluoride
0.5 mmol/L, aprotinin 0.2 kU/L, and dithiothreitol 2 mmol/L, using
a Polytron. The homogenates were centrifuged at 100 000×g
at 4 oC for 60 min . The resulting supernatant was
a portion of the cytosol (S1) fraction, and the pellet was resuspended
in the homogenizing buffer and centrifuged again at 100 000×g
at 4 oC for 60 min. The resulting supernatant (S2)
was combined with the S1 fraction, and the combination was used
for analyses as the "cytosol" fraction. The concentration
of protein in these two fractions was determined using the procedure
of Lowry et al.
Equal volumes of protein samples (40 mg of protein) and gel loading
solution (Tris-HCl 50 mmol/L pH 6.8, b-mercaptoethanol 4%, sodium
dodecyl sulfate 1%, glycerol 40%, and a trace amount of bromphenol
blue) were mixed, and the samples were boiled for 3 min and kept
on ice for 10 min. The samples were placed onto 12.5% (w/v) acrylamide
gel using the Mini Protean gel apparatus (CLP, USA). The gels were
electrophoresed using Tris base 25 mmol/L, glycine 192 mmol/L, and
sodium dodecyl sulfate 0.1% (w/v) at 150 V. The proteins were subsequently
transferred electrophoretically to an ECL nitrocellulose membrane
(Amersham) using the CLP semi-dry Electroblotter(CLP, USA) at 150
mA constant current. Membranes were washed with TBST buffer (Tris
base 10 mmol/L, NaCl 0.15 mol/L, and Tween-20 0.05%) for 10 min.
The blots were blocked by incubating them with 5% (wt/vol) powdered
non-fat milk in TBST buffer, Nonidet P-40 2 mL, and sodium dodecyl
sulfate 0.02% (wt/vol) (pH 8.0). Thereafter, the blots were incubated
overnight with the primary anti-5-LO antibody (rabbit polyclonal;
cayman chemical, Ann Arbor, MI, USA) at a dilution of 1:1000. The
blots were then washed with TBST buffer, incubated with horseradish
peroxidase-linked secondary antibody (anti-rabbit IgG; 1:3000) for
4 h at room temperature, and processed with the ECL kit. The blots
were then washed with TBST and exposed to ECL film. The UVP Image
Analyzer was used to measure and analyze the band intensity.
Statistical analysis Data were presented as mean±SD.
Statistical difference was determined using one-way analysis of
variance followed by the Student-Newman-Keuls method for multiple
comparisons between groups. All statistical calculations were performed
using a Sigmastat statistical package.
Results
Antigen-induced changes of IL-4 and IFN-¦Ã, TNF-¦Á, and NO in
BALF and cerebral cortex homogenate Amounts of IL-4, TNF-¦Á,
and NO from BALF and cerebral cortex homogenates in antigen-challenged
rats were markedly higher compared with samples from normal rats
(P<0.05). In contrast, amounts of IFN-¦Ã in antigen-challenged
rats were less (BALF P<0.05, cerebral cortex homogenates
P<0.01). Therefore, the IFN-¦Ã/IL-4 ratio was lowered.
DXM-treated rats had less IL-4, TNF-¦Á, and NO amounts in cerebral
cortex homogenates and higher IFN-¦Ã amounts compared with that of
model rats. The down-regulation of IFN-¦Ã/IL-4 ratio in antigen-challenged
rats was recovered by DXM (BALF P<0.05 and cerebral cortex
homogenates P<0.01). Ketotifen had no effect on the amount
of IL-4, IFN-¦Ã, and NO in BALF and cerebral cortex homogenates,
but inhibited the TNF-¦Á both in BALF and cerebral cortex homogenates
in model rats (P<0.05) (Table 1).
5-LO and LTA4-H mRNA expression in lung tissue and
cerebral cortex Using GAPDH as the internal control, the band
intensity of 5-LO and LTA4-H mRNA is shown in Figures
1, 2. The expression of 5-LO and LTA4-H mRNA in asthma
rats were significantly higher than that in the control rats (P<0.05).
Both DXM and ketotifen inhibited the 5-LO and LTA4-H
mRNA expression of lung and cerebral cortex in the asthma rats (Figures
1, 2).
5-LO protein content Both 5-LO mRNA expression and the amount
of cytosol 5-LO-immunoreactive protein in both cerebral cortex and
lung tissue increased in the asthma rats compared with the control
rats (P<0.05 and P<0.01 respectively; Figure
3). Both DXM and ketotifen significantly decreased the 5-LO protein
level in cytosol of asthma rats (P<0.05).
Discussion
The first step in the production of all LTs, the oxygenation
of arachidonic acid (AA) to form 5-hydroperoxyeicosatetranoic
acid and the immediate dehydration of this unstable intermediate
to LTA4, is carried out by 5-LO. Metabolism of
LTA4 by LTA4 hydrolase (LTA4-H)
results in the production of the potent chemoattractant LTB4.
Alternatively, LTA4 can be conjugated with glutathione
by LTC4 synthase (LTC4-S) to produce
LTC4 and its metabolites LTD4 and
E4, collectively referred to as the cysteinyl LTs[6].
The actions of 5-LO metabolites and Th1/Th2 paradigm in the pathogenesis
of asthma and allergic disease have been understood[7,8].
In the present research, IL-4 level, the expression of 5-LO and
LTA4-H mRNA, and 5-LO protein are simultaneously increased
in BALF and the cerebral cortex in asthma rats. We postulate that
the increased expression of 5-LO pathway enzymes is partly due to
the overproduction of proinflammatory cytokines, as heterogeneous
profiles of eicosanoid generation would be determined by the absence
or presence of additional local factors, particularly the Th2 cytokines
that associate with mucosal surfaces in allergic diseases. Previous
literature has shown that Th2 cytokines, thought to favor asthma
and allergic diseases, upregulated LT synthesis. Recent findings
showed that IL-4, IL-5, and IL-13 also upregulated cysteinyl LT1
receptor expression[9]. IL-4 priming dramatically
induced the steady-state expression of LTC4-S[10],
and IL-5 supported the localization of 5-LO to the nucleus of human
mast cells in vivo[11]. With immunologic
disorders, inflammatory mediators interacted with one another, creating
upregulatory cascades by various feedback mechanisms[12].
Conversely, the regulation of cytokine expression by LTs has also
been explored: cysteinyl LTs upregulated Th2 cytokine expression
and decreased Th1 cytokine expression, favoring an allergic phenotype[13].
LTB4 stimulated monocytes and T cell to produce IL-1,
TNF-¦Á, and IL-5[14]. However, it is unclear
how it influences pulmonary function during the increase of expression
of 5-LO, LTA4-H mRNA, and LTB4 level in the
cerebral cortex. Recently, our group found exogenous LTB4,
by intercerebro-ventricularly injection, inhibited antigen-induced
increase of lung resistance and decreased lung compliance in sensitized
rats, and reduced antigen-induced eosinophils infiltration of airway
in the sensitized mice (unpublished data). The results suggest the
increase of 5-LO mRNA expression and metabolites in inflammatory
status of asthma may be a negative feedback to relieve asthmatic
inflammation.
Glucocorticoids are the most effective drugs to inhibit inflammatory
reaction in asthma, but whether they are also able to inhibit the
5-LO pathway enzyme expression in sensitized rats after antigen
challenge is still unclear. In our previous experiment, the effect
of DXM in decreasing LTB4 and IL-4 content was found
to decrease the number of inflammatory cells in cerebral cortex
and lung tissue of sensitized rats[2,3]. In this study,
administration of DXM (0.5 mg/kg, ip) before each challenge fully
inhibited the 5-LO and LTA4-H mRNA expression. Conversely,
Uz et al[15] found that DXM stimulated 5-LO mRNA
expression in the brain of normal rats. These data suggest that
glucocorticoid may be at least partly effective in regulating the
metabolites of 5-LO pathways in the brain.
It is known that TNF-¦Á is released in allergic responses from both
mast cells and macrophages via IgE-dependent mechanisms, and elevated
levels have been demonstrated in the BALF of asthmatic subjects
undergoing allergen challenge[16]. In the present research,
we found a coincidental increase of TNF-¦Á between the cerebral cortex
and lung tissue in asthma rats, which could be inhibited by DXM
and ketotifen. Ketotifen, as the mast cell membrane stabilizer and
H1 receptor antagonist, can inhibit the release of mediators
from inflammatory cells. Our previous study demonstrated that ketotifen
inhibited the increase of LTB4 level in the cerebral
cortex and BALF in asthma rats[3]. In this study, ketotifen
did not inhibit the downregulation of IFN-¦Ã and upregulation of
IL-4 but decreased 5-LO and LTA4-H mRNA expression and
TNF-¦Á level in asthma rats. Canetti et al[17]
found that using a neutralizing anti-TNF-¦Á antibody could block
IL-18 induced LTB4 production, and Spanbroek et al[18]
found granulocyte-macrophage colony-stimulating factor plus TNF-¦Á
promoted dendritic cells differentiation and induced a strong rise
in 5-LO expression. The results suggest that ketotifen may not influence
the Th1/Th2 paradigm but inhibits the release of mediators from
mast cells. TNF-¦Á may be also one of the proinflammatory mediators
of regulating 5-LO and LTA4-H mRNA expression.
NO can inhibit the secretion of IL-2 and IFN-¦Ã by Th1
cells but has no effect on IL-4 production by Th2 cells.
Thus, NO seems to exert a self-regulatory effect on Th1 cells
implicated in immunopathology. NO derived from airway
epithelial cells, macrophages, and Th1 cells plays an
important role in amplifying and perpetuating the Th2 cell-mediated
inflammatory response, both in allergic and non-allergic asthma[19].
Low concentrations of reactive oxygen intermediates in combination
with NO were additive in suppressing 5-LO enzyme activity, possibly
through peroxynitrite generation. The ability of peroxynitrite
to suppress LT synthesis is associated with its ability to
cause nitrotyrosination and S-nitrosylation of the 5-LO enzyme[20].
Contrary to the inhibitory effect of DXM on NO content in this research,
ketotifen did not inhibit the increased NO level in the asthma rats.
Therefore, the regulatory effect of NO on 5-LO and LTA4-H
requires elucidating.
In the present research, the correlative increase of 5-LO pathway
and proinflammatory mediators between cerebral cortex and lung tissue
were shown in sensitized rats after repeated antigen challenge.
We postulate that an increase of 5-LO pathway expression and pro-inflammatory
mediators of the brain may have regulatory effects on pulmonary
inflammation of asthma.
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