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Department of Oriental Pharmacy, College of Pharmacy, Wonkwang University, Iksan, Chonbuk, South Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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7-fold compared with that of basal cells.
The antisense TGF-ß1 oligonucleotides also had a significant
inhibitory effect on anti-DNP IgE-induced TNF-
release from mast
cells. In situ hybridization analysis showed that the PCA reaction
sites treated with antisense TGF-ß1 oligonucleotides exhibited no
detectable levels of TGF-ß1 and L-histidine decarboxylase
mRNA after anti-DNP IgE stimulation, whereas the PCA reaction sites
treated with sense TGF-ß1 oligonucleotides possessed significant
amounts of their mRNA. Additionally, neutralizing Ab to TGF-ß1
blocked the PCA reaction significantly, but its Ab did not inhibit
peritoneal mast cell-released histamine upon treatment with
anti-DNP IgE. Our results suggest that TGF-ß1 is critical to the
development of IgE-dependent anaphylaxis
reactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-1 (I)
collagen mRNA in dermal fibroblast and that mast cell-derived TGF-ß1
importantly contribute to this effect.
Therefore, we investigated this possibility using experimental
IgE-dependent anaphylactic reaction system in vivo and in vitro. Among
the preformed and newly synthesized inflammatory substances released on
the degranulation of mast cells, histamine remains the best
characterized and most potent vasoactive mediator implicated in the
acute phase of anaphylaxis (10). L-histidine decarboxylase
(HDC)3 catalyzes the formation
of histamine from its precursor, histidine, in a single step. Activated
mast cells also produce the multipotent cytokine TNF- (11). The
secretory response of mast cells can be induced by aggregation of their
cell surface-specific receptors for IgE Ab by the corresponding Ag
(12, 13, 14). It has been established that the anti-IgE Ab induces
passive cutaneous anaphylaxis (PCA) reaction (15).
In this paper, we have evaluated the role for mast cell-derived
TGF-ß1 in IgE-dependent anaphylaxis. Given the importance of TNF-
in inflammation (16), cytotoxicity (17), and immune function (18), we
also investigated the influence of antisense TGF-ß1 oligonucleotides
on TNF- release from mast cells. Our findings demonstrate that
IgE-dependent anaphylaxis can induce an increase in steady state levels
of HDC mRNA in mast cells and that mast cell-derived TGF-ß1
importantly contribute to this effect.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free Wistar rats were purchased from the Dae-Han Experimental Animal Center (Eumsung, South Korea), and the animals were maintained at the College of Pharmacy, Wonkwang University (South Korea). The animals were housed five to ten per cage in a laminar air flow system maintained under a temperature of 22 ± 1°C and relative humidity of 55 ± 10% throughout the study.
Reagents
Anti-DNP IgE, DNP-human serum albumin (HSA), and metrizamide
were purchased from Sigma (St. Louis, MO). FCS was purchased from Life
Technologies (Grand Island, NY). rTNF- (1 x 105
U/ml) and rabbit anti-murine TNF- and TGF-ß1 Ab were purchased
from Genzyme (Munchen, Germany). TGF-ß1 was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Phosphatase-labeled anti-rabbit IgG
was purchased from Serotec (Oxford, U.K.).
Synthetic oligonucleotides
Modified (phosphorothioate) antisense and sense oligonucleotides were synthesized on an Applied Biosystems (Foster City, CA) model 394 high-throughput DNA synthesizer. The oligonucleotides were lyophilized, resuspended in PBS, and quantified by spectrophotometry. Based on the already-known murine TGF-ß1 sequence (19), 25-mer TGF-ß1 antisense oligonucleotides (5'-CAGCCCCGAGGGCGGCATGGGGGAG-3') complementary to sequences surrounding the AUG codon of TGF-ß1 were synthesized. Sense oligonucleotides (5'-CTCCCCCATGCCGCCCTCGGGGCTG-3') specific of murine TGF-ß1 were used as a control. The antisense TGF-ß1 oligonucleotides or the corresponding sense TGF-ß1 oligonucleotides were diluted in PBS solution and kept at 4°C.
PCA
An IgE-dependent skin reaction was generated by sensitizing the skin with an intradermal injection of anti-DNP IgE followed by an injection 48 h later of DNP-HSA into the rat’s tail vein. The rats were injected intradermally with 0.5 µg (50 µl) of anti-DNP IgE into each of four dorsal skin sites that had been shaved 48 h earlier. The sites were outlined with a water-insoluble red marker. After 48 h, each rat received an injection of 1 mg of DNP-HSA in PBS containing 4% Evans blue (1:4) through the tail vein. The TGF-ß1 oligonucleotides and TGF-ß1-neutralizing Ab (Santa Cruz Biotechnology) were added topically 30 min before challenge with DNP-HSA. The anti-DNP IgE and DNP-HSA were diluted in PBS. One hour after the challenge, the rats were killed, and the dorsal skin was removed for measurement of pigment area. The amount of dye was then determined colorimetrically after extraction with 1 ml of 1.0 N KOH and 9 ml of mixture of acetone and phosphoric acid (5:13) in accord with the method of Katayama et al. (20). The absorbant intensity of the extraction was measured at 620 nm using a spectrophotometer, and the amount of dye was calculated with the Evans blue measuring line.
Preparation of rat peritoneal mast cells
Peritoneal mast cells were isolated as previously described
(21). In brief, rats were anesthetized by ether, and 20 ml of Tyrode
buffer B (NaCl, glucose, NaHCO3, KCl,
NaH2PO4) containing 0.1% gelatin (Sigma) was
injected into the peritoneal cavity, and the abdomen was gently
massaged for 90 s. The peritoneal cavity was carefully opened, and
the fluid containing peritoneal cells was aspirated using a Pasteur
pipette. Thereafter, the peritoneal cells were sedimented at 150
x g for 10 min at room temperature and resuspended in
Tyrode buffer B. Mast cells were separated from major contaminants of
rat peritoneal cells, i.e. macrophages and small lymphocytes, according
to the method described by Yurt et al. (22). In brief, peritoneal cells
suspended in 1 ml Tyrode buffer B were layered on 2 ml of 22.5% w/v
metrizamide (density, 1.120 g/ml) (Sigma) and centrifuged at room
temperature for 15 min at 400 x g. The cells remaining
at the buffer-metrizamide interface were aspirated and discarded; the
cells in the pellet were washed and resuspended in 1 ml Tyrode buffer A
containing calcium. Mast cell preparations were 95% pure as
assessed by toluidine blue staining. More than 97% of cells were
viable as judged by trypan blue uptake.
Assay of histamine release
Mast cell suspensions (1 x 106 cells/ml) were sensitized with anti-DNP IgE (10 µg/ml) for 6 h. The cells were preincubated with the TGF-ß1 oligonucleotides at 37°C for 10 min before the challenge with DNP-HSA (1 µg/ml). The cells were separated from the released histamine by centrifugation at 400 x g for 5 min at 4°C. Residual histamine in cells was released by disrupting the cells with perchloric acid and centrifugation at 400 x g for 5 min at 4°C. Histamine content was measured by the o-phthalaldehyde spectroflurometric procedure of Shore et al. (23). The fluorescent intensity was measured at 438 nm (excitation at 353 nm) in a spectrofluorometer.
The inhibition percentage of histamine release was calculated by using the following equation: % inhibition = {[(histamine release without TGF-ß1 oligonucleotides - histamine release with TGF-ß1 oligonucleotides) x 100]/[histamine release without TGF-ß1 oligonucleotides]}.
cAMP assay
The cAMP level was measured according to the method of Peachell et al. (24). In brief, purified mast cells were resuspended in prewarmed (37°C) Tyrode buffer A. Typically, an aliquot of cells (1 x 106 cells) was added to an equivalent volume (50 µl) of prewarmed buffer containing the drug in an Eppendorf tube. The reaction was allowed to proceed for discrete time intervals, terminated by the addition of ice-cold acidified ethanol (0.9 ml of 86% ethanol/1 M HCl, 99:1) with brief vigorous vortexing, and then snap frozen in liquid nitrogen. The sample was later thawed and vortexed, then the debris was sedimented in a centrifuge (400 x g at 4°C for 5 min), and an aliquot (0.9 ml) of the supernatant was removed and evaporated to dryness under a reduced pressure. The dried sample was reconstituted in assay buffer (150 - 200 µl) and stored frozen. The cAMP level was determined by enzyme immunoassay, using a commercial kit (Amersham International).
RNA extraction and Northern blot analysis
Total RNA was prepared by using the modified LiCl-urea method
(25), electrophoresis in 1.2% agarose-formaldehyde gels, and
transferred to nylon membranes by capillary action in 20x SSC (1x
SSC = 0.15 M NaCl and 0.015 M sodium citrate (pH 7.2)). After
prehybridization, the filters were hybridized with random
[-32P]dCTP-labeled probes having specific activity of
1 to x 108 cpm/µg in 10% dextran sulfate, 50%
formamide, 4x SSC, 1x Denhardt’s solution, and 10 µg/ml salmon
sperm DNA for 24 h at 42°C. Then the filters were washed, dried,
and examined by autoradiography.
Assay of TNF- and TGF-ß1 release
TNF- and TGF-ß1 secretion were measured by a modified
ELISA, as described (26). The ELISA was devised by coating 96-well
plates of murine monoclonal Ab with specificity for murine TNF- and
TGF-ß1. Before use and between subsequent steps in the assay, coated
plates were washed twice with PBS containing 0.05% Tween 20 and twice
with PBS alone. All reagents used in this assay were incubated for
1 h at room temperature with coated wells. For the standard curve,
rTNF- and rTGF-ß1 were added to serum previously determined to be
negative for endogenous TNF- and TGF-ß1. After exposure to medium,
assay plates were exposed sequentially to rabbit anti-TNF- and
TGF-ß1, phosphatase-conjugated goat anti-rabbit IgG, and
p-nitrophenyl phosphate. OD readings were made within 10 min
of addition of the substrate on a Titertek Multiscan (Flow
Laboratories, North Ryde, Australia) with a 405-nm filter. Appropriate
specificity controls were included.
In situ hybridization
Digoxigenin-labeled single-strand RNA probes were prepared with
the DIG RNA Labeling Kit (Boehringer Mannheim, Mannheim, Germany),
according to the manufacturer’s instructions. To generate a murine
TGF-ß1-specific probe, a SmaI-digested fragment of the
TGF-ß1 cDNA clone (19) was subcloned into the pBluescript I PKS(-)
plasmid. The HDC probe used a PstI-digested cDNA insert from
the HDC cDNA clone kindly provided Dr. A. Ichikawa (Kyoto University,
Kyoto, Japan) (27). These plasmids were either transcribed with T3 RNA
polymerase to generate an antisense (cRNA) probe or transcribed with T7
RNA polymerase to generate a sense probe. Hybridization was conducted
as previously described (28), with minor modifications. All solutions
used were treated with 0.02% diethylpyrocarbonate (Sigma) and
autocalved, and all glassware was baked at 180°C for >3 h to
inactivate RNases. Before hybridization, sections were fixed with
freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (pH
7.4) for 20 min. Thereafter, they were treated with 0.2 N HCl to
inactivate endogenous alkaline phosphatase activity and acetylated with
0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min.
This was followed by rinsing twice in 0.1 M PBS for 5 min and
dehydration through a graded (70, 80, 90, 95, and 99.5%) ethanol
series; the slides were then air-dried. The hybridization solution
contained 50% deionized formamide, 10% dextran sulfate, 1x
Denhardt’s solution, 600 mM NaCl, 10 mM DTT, 0.25% SDS, 250
µg/ml of Escherichia coli transfer RNA, and 0.5
µg/ml of digoxigenin-labeled RNA probe. Hybridization solution (50
µl) was placed on each section, which was covered with parafilm and
incubated at 50°C for 16 h in a moisture chamber. After
hybridization, parafilm was dislodged by briefly washing with 5x SSC
(1x SSC = 0.15 M NaCl, 0.015 M sodium citrate) and with 50%
formamide and 2x SSC for 30 min at 50°C. RNase A digestion (10
µg/ml) proceeded at 37°C for 30 min. The sections were washed twice
with 2x SSC and 0.2x SSC for 15 min each time at 50°C. The
hybridized digoxigenin-labeled probe was detected with a Nucleic Acid
Detection Kit (Boehringer Mannheim), according to the manufacturer’s
instructions. After the color reaction, the slides were rinsed in 10 mM
Tris-HCl (pH 8.0) and 1 mM EDTA. Controls included: 1) hybridization
with the sense (mRNA) probe, 2) RNase A treatment (20 µg/ml) before
hybridization for 30 min at 37°C, and 3) use of neither antisense RNA
probe nor antidigoxigenin Ab. None of the three controls showed
positive signals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the contribution of TGF-ß1 in the anaphylactic
reaction, we initially ascertained the effect of antisense TGF-ß1
oligonucleotides on the in vivo model of IgE-dependent PCA reaction in
rats. Skin extravasation is induced by a local injection of
anti-DNP IgE followed by an i.v. antigenic challenge. The addition
of antisense TGF-ß1 oligonucleotides dose-dependently inhibited the
PCA reaction (Fig. 1
, A-C). As
a control, the corresponding sense TGF-ß1 oligonucleotides were
studied comparatively at the same concentration. The addition of sense
TGF-ß1 oligonucleotides did not affect the in vivo PCA reaction
significantly. As shown in Fig. 1D, antisense TGF-ß1
oligonucleotides inhibited histamine release from the peritoneal mast
cells activated by anti-DNP IgE in a concentration-dependent
fashion, but did not significantly affect the mast cell numbers or
viability, indicating that the inhibitory effect on histamine release
was not due to a toxic effect on the cells. Sense TGF-ß1
oligonucleotides had no significant effect. Data in Fig. 2 show the effect of the antisense
TGF-ß1 oligonucleotides on the cAMP level of peritoneal mast cells.
When peritoneal mast cells were incubated with antisense TGF-ß1
oligonucleotides at the concentration of 100 µg/ml, the cAMP content
significantly increased. It peaked 3 min after antisense TGF-ß1
oligonucleotides were added, and then decreased to basal value about 10
min later. Antisense TGF-ß1 oligonucleotides also inhibited
anti-DNP IgE-mediated release of TGF-ß1 and TNF- from
peritoneal mast cells (Table I and Table II).
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Biologically active TGF-ß results after dissociation from the
latency-associated peptide (29). TGF-ß can exert a variety of
effects, depending on the maturity of the target cell and the
physiological condition. We next ascertained the effect of antisense
TGF-ß1 oligonucleotides in suppressing TGF-ß1 mRNA levels in the
rat peritoneal mast cells. Northern blot analysis shows that activated
peritoneal mast cells treated with antisense oligonucleotides exhibit
detectable levels of TGF-ß1 mRNA, whereas the cells treated with
sense oligonucleotides possess significant amounts of TGF-ß1 mRNA
(Fig. 3). To gain further insight into
the biological function of TGF-ß1 in vivo, we next assessed whether
IgE-dependent mast cell activation was associated with augmentation of
TGF-ß1 gene expression by using in situ hybridization (28) to search
for TGF-ß1 mRNA expression after PCA reactions in the skins of Wistar
rats (Fig. 4). TGF-ß1 mRNA was induced
in mast cells at the PCA reaction sites (Fig. 4B). The
intensity of the signals then began to wane until, by 48 h after
Ag challenge, labeling was back to baseline levels. We then determined
the effect of antisense TGF-ß1 oligonucleotides on PCA reaction sites
in 16 rats who were treated with PBS containing antisense
oligonucleotides (n = 7), sense oligonucleotides
(n = 3), no oligonucleotides (n = 3),
or were untreated (n = 3). In situ hybridization
analysis revealed that expression is blocked with antisense TGF-ß1
oligonucleotides, whereas a similar expression level was observed in
the other treatment (Fig. 4D). Although we also evaluated
the expression of TGF-ß2 and -ß3, no detectable amounts of
these genes were observed in in situ hybridizations (data not shown).
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HDC catalyzes the formation of histamine from its precursor,
histidine, in a single step. Synthesis of histamine was associated with
an increase in the expression of HDC mRNA after stimulation of mast
cells (21). The expression of HDC mRNA was up-regulated after PCA
reaction, and antisense TGF-ß1 oligonucleotides could block the
expression of HDC mRNA (Fig. 5).
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We finally examined the effect of TGF-ß1-neutralizing Ab in
IgE-dependent anaphylaxis reactions. In each animal, control group was
treated with an irrelevant Ab (rabbit IgG), and experimental group was
treated with TGF-ß1-neutralizing Ab. TGF-ß1-neutralizing Ab (100
ng/site) significantly inhibited to 28.5% PCA reaction activated by
anti-DNP IgE (Fig. 6,
A-C). TGF-ß1-neutralizing Ab did not inhibit the histamine
release from the peritoneal mast cells (Fig. 6D) and the
cAMP level of peritoneal mast cells (data not shown).
TGF-ß1-neutralizing Ab also did not inhibit the anti-DNP
IgE-mediated TNF- production (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7-fold, in comparison with that of basal cells (Fig. 2
).
The mode of the action of antisense TGF-ß1 oligonucleotides is likely
related to the prevention of calcium release from the calcium store of
mast cells, due to elevation of the intracellular cAMP level by
inhibition of the cAMP phosphodiesterase. Our data showed that
antisense TGF-ß1 oligonucleotides inhibited anti-DNP IgE-induced
TNF- production (Table I). The effect of antisense TGF-ß1
oligonucleotides on mast cell cytokine production in vivo and the
relative importance of mast cells as a source of TNF- during
inflammatory and immune responses are important areas for future
studies. The result of our in vivo experiments demonstrated that
induction of TGF-ß1 and HDC gene expression occurred in skin that was
challenged with specific Ag, that this change, at least at the earliest
stages of the response, did not reflect the infiltration of
inflammatory cells into the tissues, and that the development of the
response was entirely dependent on mast cells. Our in vivo studies
showed that the increase in TGF-ß1 mRNA occurred at PCA sites. This
result indicates that TGF-ß1 mRNA-positive mast cells were indeed
resident cells of the skin and that the initial up-regulation of
TGF-ß1 mRNA levels did depend on the activation of mast cells. Based
on these findings, we propose that TGF-ß1 represent mediators
that can contribute to the ability of activated mast cells to the
development of clinically or histologically significant tissue
fibrosis. Tissue fibrosis is a feature of chronic allergic responses,
such as long-term allergic asthma or atopic dermatitis, rather than
isolated anaphylaxis responses (8). Therefore, TGF-ß1 is likely to
facilitate both acute inflammatory and chronic changes within this
tissue. Accordingly, it will be of interest to develop model systems
that permit evaluation of the effects of persistent mast cell
activation on tissue levels of the extracellular matrix molecules.
This study is a direct proof of the effectiveness of the antisense
TGF-ß1 approach in specifically down-regulating TGF-ß1 mRNA
expression. We demonstrated by in situ hybridization that TGF-ß1
mRNA, as well as HDC mRNA, is decreased. Thus it seems as if the
administration of the antisense TGF-ß1 oligonucleotides in the
sensitized sites may result in the down-regulation of various mRNAs. In
addition, based on the in situ part of the figures (Fig. 4, B and D), it appears that TGF-ß1 message is
still present in other cell types. Therefore, it is not possible to
conclude that the effects of the antisense TGF-ß1 oligonucleotides on
the PCA reaction in vivo were specifically due to effects on mast
cells. Although it is not clear why mast cell message would be almost
totally abrogated, but TGF-ß1 message in other cell types in the same
area minimally affected, this may suggest that anti-DNP IgE induced
mast cell activation is selectively blocked by antisense TGF-ß1
oligonucleotides. The experiment of treating cells in vitro with the
antisense TGF-ß1 oligonucleotides and demonstrating a direct effect
on the TGF-ß1 mRNA and protein is critically important to both the
premise and conclusion of this study. Thus, a detailed in vitro
analysis is indispensable to interpretation of in vivo results in
future study. Furthermore, this study showed that antisense TGF-ß1
oligonucleotide treatment of mast cells or sensitized skin sites may
result in a generalized down-regulation of mast cell responses since
PCA is inhibited, TGF-ß1 and HDC mRNA levels are reduced, and
histamine and TNF- secretion are inhibited. Because the responses
are measured after an IgE-dependent stimulus, it is difficult to
understand how antisense TGF-ß1 oligonucleotide treatment for 10 min
at 37°C could inhibit the activation of Fc
RI and subsequent
events. Therefore, we need to do further in vitro experiments on signal
transduction cascade of mast cell activation via FcRI that can be
augmented by TGF-ß1 or blocked by Abs to TGF-ß1. In particular, the
in vitro experiments with neutralizing Ab to TGF-ß1 did not result in
inhibition of histamine release, while the in vivo experiments with the
same neutralizing Ab inhibited PCA. This would suggest an indirect
effect of the neutralizing Ab that is not TGF-ß1-dependent. In
addition, the ineffectiveness of Ab with isolated mast cells is
indicated because the protein should not enter intact cells.
Nevertheless, that an Ab applied topically would penetrate is rather
surprising. It is assumed that it may be due to skin injury by shaving
before the treatment of Ab, but it needs more study for elucidating
complex biological properties. Taken together, the results presented in
this paper are difficult to resolve with a previous demonstration by
Bissonnette et al. (31) that TGF-ß1 inhibited release of TNF- by
mast cells instead of stimulating it. A convincing demonstration of a
role for TGF-ß1 in anaphylaxis could be achieved by the use of
available TGF-ß1 knockout mice. While these animals have a short life
span, they can be manipulated to have an extended life span,
thus, bone marrow-derived mast cells can be obtained. Additional
experiments that are critical to interpretation of these series of
experiments are required. Our data suggest a critical link between
TGF-ß1 and mast cells by the finding that mast-cell-derived TGF-ß1
serves as an essential anaphylactic factor. Our findings also suggest
an important new approach to the control of IgE-dependent anaphylactic
reaction.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hyung-Min Kim, Department of Oriental Pharmacy, College of Pharmacy, Wonkwang University, Iksan, Chonbuk, 570-749, South Korea. E-mail address:
3 Abbreviations used in this paper: HDC, L-histidine decarboxylase; PCA, passive cutaneous anaphylaxis; HSA, human serum albumin.
Received for publication August 18, 1998. Accepted for publication January 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI: role for mast cell-derived transforming growth factor ß and tumor necrosis factor . J. Exp. Med. 180:2027./cachectin. Nature 346:274.[Medline]
RI): role of cytoplasmic domains. J. Biol. Chem. 266:22613. in disease states and inflammation. Crit. Care Med. 21:S447.[Medline]
. FASEB J. 4:3215.[Abstract]
: a multifaceted peptide hormone. Crit. Rev. Immunol. 9:21.[Medline]
gene. Dev. Brain Res. 70:239.[Medline]
from mast cells through an autocrine pathway. Am. J. Respir. Cell Mol. Biol. 16:275.[Abstract]
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, Sense TGF-ß1
oligonucleotides. Value is the mean ± SE of seven independent
experiments. *, p < 0.05; **,
p < 0.01, significantly different from saline
value.
