<title>1. INTRODUCTION</title> <p>Allergic rhinitis (AR) is well accepted to be a symptomatic
disease of the nasal mucosa caused by an IgE-mediated allergic inflammation,
and characterized by nasal itching, sneezing, water rhinorrhea, and nasal
obstruction, which makes breathing through the nose difficult [##REF##11291780##1##]. These
clinical symptoms are also well known to be mediated by several factors such as
histamine, prostaglandins, and other inflammatory mediators (e.g., inflammatory
cytokines) secreted from activated inflammatory cells including eosinophils,
mast cells, and T cells in the local of inflammation [##REF##11291780##1##]. In addition to these
classical immune responses, structural changes within the nasal walls have also
been reported in patients with allergic rhinitis. These structural changes
include epithelial disruption, mucus gland hypertrophy, mucosal myofibroblast
transformation, and increased matrix protein deposition [##REF##14656347##2##, ##REF##14713906##3##]. These cellular
changes are now called tissue remodeling and two groups of proteins matrix
metalloproteinases (MMPs) and their counter-regulatory inhibitors, TIMPs, are
generally accepted to be important factors for tissue remodeling [##REF##15663564##4##].</p> <p>Recently, there is increasing evidence that angiogenesis
plays an important role in both the development of inflammation and in the
pathophysiology of tissue remodeling during allergic responses [##REF##14656347##2##, ##REF##15643343##5##, ##REF##17651148##6##].
Numerous numbers of inducers of angiogenesis have been identified, including vascular
endothelial growth factor (VEGF), angiogenin, transforming growth factor (TGF),
tumor necrosis factor-<italic>α</italic> (TNF) and
interleukin (IL)-8, and others [##REF##17651148##6##, ##REF##16159620##7##]. Some experimental evidence strongly
suggests that in inflammation, infiltrating inflammatory cells and some
resident cells are the producers of the angiogenic factors. Human neutrophils
[##REF##9354686##8##] and T-lymphocytes [##REF##8841458##9##] synthesize and secret the angiogenic factors such as
VEGF and IL-8. Peripheral blood eosinophils were found to secrete the factors
when stimulated with granulocyte-macrophage colony stimulating factor and IL-5 in
vitro [##REF##9224211##10##]. Fibroblasts, as resident cells, are also a demonstrated rich source
of the angiogenic factors [##REF##10699968##11##]. Among these cells, mast cells and eosinophils
have been highlighted as the effector cells in angiogenesis during allergic
inflammation [##REF##16159620##7##, ##REF##15618019##12##]. Although recent
researches have focused on the ability of antihistamines, which are the most
important agent in the treatment of allergic diseases including AR, to modulate
the release of inflammatory cytokines from mast cells and eosinophils, there is
little information regarding the effects of antihistamines on angiogenesis. The
present study, therefore, was undertaken to examine the influence of epinastine
hydrochloride (EP), the most famous antihistamine in Japan, on keratinocyte-derived
chemokine (KC), TNF, and VEGF that are known to be major factors affecting
angiogenesis [##REF##12076253##13##] in murine mast cells in IgE-dependent manner.</p>
<title>2. MATERIALS AND METHODS</title> <title>2.1. Mice</title> <p>Specific pathogen-free
BALB/c male mice were purchased from Charles River Japan Inc. (Atsugi, Japan).</p> <title>2.2. Agents</title> <p>EP was kindly donated by
Nihon Boehringer Ingelheim Co. Ltd. (Tokyo, Japan) as a
preservative-free pure powder. This was dissolved in RPMI-1640 medium (SIGMA-ALDRICH Inc., St Louis, MO, USA)
supplemented with 10% heat inactivated fetal calf serum from GIBCO BRL (Gaithersburg,
Md, USA; RPMI-FCS) at a concentration of 10 mg/mL, sterilized by passing
through 0.2 <italic>μ</italic>m filter and then diluted with RPMI-FCS at appropriate concentrations for experiments.</p> <title>2.3. Preparation of mouse IgE</title> <p>BALB/c mice were injected
intraperitoneally twice with a 2-week interval with 5.0 mg of ovalbumin from WAKO Pure Chemicals (OVA;
Osaka, Japan) absorbed with 0.5 mg of alum in a volume of 0.5 mL. After one week, blood was obtained by
cardiac puncture and OVA specific IgE was purified with KAPTIV-AE from Tecnogen
S.C.p.A. (Monte Verna, Italy)
according to the manufacturer's instructions. The protein concentration of the extracted
solution was measured with protein assay kit from Bio-Rad Laboratories (Hercules, Calif, USA) 
and adjusted to 1.0 mg/mL with RPMI-FCS.</p> <title>2.4. Preparation of mast cells</title> <p>Mouse peritoneal mast cells were isolated as previously described [##REF##12076253##13##]. Briefly, mice
were anesthetized with diethyl ether and exanguinated by decapitation. The
peritoneal cavity was rinsed with 10 mL of heparinized (10 IU/mL) calcium and
magnesium-free Hank's solution (HBSS). The fluid was collected, centrifuged at
150 g for 15 minutes at 4°C. The pelleted cells were resuspended in
HBSS containing 0.1% bovine serum albumin and submitted to a continuous
isotonic Percoll gradient (72%) for mast cell isolation. Purified mast cells
were resuspended in RPMI-FCS. The cell purity (&gt;96%) and viability (&gt;98%)
were evaluated by alcian blue and trypan blue exclusion staining, respectively.</p> <title>2.5. Sensitization and treatment of mast cells</title> <p>To sensitize mast cells with IgE, 1 × 10<sup>6</sup>, cells were incubated with 500 ng/mL of
OVA-specific IgE for 30 minutes at 37°C, washed, and resuspended in
RPMI-FCS. The sensitized mast cells (5 × 10<sup>5</sup> cells/mL) were
stimulated with 10 ng/mL OVA for 4 hours in the presence of various concentrations
of EP in a total volume of 2.0 mL. The culture supernatants were collected
after centrifugation of cell suspension at 150 g for 15 minutes at 4°C
and stored at −40°C
prior to assay for factors. To obtain water-soluble intracellular contents, the pelleted cells suspended in 2.0 mL HBSS were then sonically disrupted in an ice cold water bath for 5 minutes
and centrifuged at 3000 g for 30 minutes at 4°C. The supernatants
were obtained and stored at −40°C
until used. In cases of examining mRNA expression, mast cells sensitized IgE were stimulated with OVA in a
similar manner for 4 hours and stored at −80°C
until used. In all experiments, EP was added to cell cultures one hour before
starting antigenic stimulation and cells were cultured in triplicate.</p> <title>2.6. Assay for factors</title> <p>KC, TNF, and VEGF levels in
culture supernatants were assayed using commercially available mouse ELISA test
kits (R &amp; D systems, Minneapolis, Minn, USA)
according to manufacturer's recommendation.</p> <title>2.7. Real-time polymerase chain reaction</title> <p>mRNA was extracted from mast cells using <italic>μ</italic>MACS mRNA
isolation kits from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany)
according to the manufacturer's instructions. The first-strand
complementary DNA (cDNA) synthesis from 1.0 <italic>μ</italic>g mRNA was
performed using the SuperScript Preamplification System for cDNA synthesis from
GIBCO BRL (Gaithersburg, Md, USA). PCR was then carried out using a GeneAmp 5700 Sequence Detection System from
Applied Biosystems (Foster City, Calif, USA). PCR mixture consisted of 2.0 <italic>μ</italic>l of
sample cDNA solution (10.0 ng/mL), 25.0 <italic>μ</italic>l of SYBR-Green Mastermix (Applied Biosystems), 0.3 <italic>μ</italic>l of both
sense and antisense primers, and distilled water to give a final volume of 50.0 <italic>μ</italic>l. The 
reaction was conducted as follows: 4 minutes at 95°C, followed by 40
cycles of 15 seconds at 95°C, and 60 seconds at 60°C. <italic>β</italic>-actin was
amplified as an internal control. mRNA levels were calculated by using the comparative parameter threshold cycle (Ct) and normalized
to <italic>β</italic>-actin. Oligonucleotide sequences of the primers used are as follows: for 
KC [##REF##17471351##14##], 5′-GCGCCTATCGCCAATGAG-3′ (sense) and 
5′-AGGGCAACACCTTCAAGCTCT-3′ (antisense); for VEGF [##REF##17659277##15##], 5′-CAGCTATTGCCGTCCGATTGAGA-3′ (sense) and 5′-TGCTGGCTTTGGGAGGTTTGAT-<italic/>
<italic/>
<italic/>3′ (antisense); 
for TNF [##REF##16288994##16##], 5′-CCTGTAGCCCACGTCGTAGC-3′ and
5′-TTGACCTCAGCGCTGAGTTG-3′; for <italic>β</italic>-actin [##REF##16288994##16##],
5′-ACCCACACTTGTGCCCATCTA-3′ (sense) and 5′-CGGAACCGCTCATTGCC-3′ (antisense).</p> <title>2.8. Statistical analysis</title> <p>The statistical significance of the data between the control and experimental
groups was analyzed by ANOVA followed by Fisher's PLSD test. <italic>P</italic> &lt; .05 was considered statistically.</p>
<title>3. RESULTS</title> <title>3.1. Suppressive activity of EP on KC, TNF, and VEGF production from mast cells</title> <p>The first set of experiments was undertaken to examine the influence of EP on KC, TNF and VEGF
production from mast cells induced by antigenic stimulation in vitro. Mast cells
(5 × 10<sup>5</sup> cells/mL) sensitized with OVA specific IgE were stimulated
with OVA in the presence of 0, 10, 20, 25, 30, and 40 ng/mL EP for 4 hours. Factor levels in culture supernatants were
assayed by ELISA. As shown in Figures ##FIG##0##1(a)## and ##FIG##0##1(b)##, 
treatment of cells with EP
at quantities lower than 20 ng/mL did not cause the suppression of the release
of both KC and TNF, which was increased by antigenic stimulation. However, EP
significantly suppressed the ability of cells to release both KC and TNF after
antigenic stimulation, when the agent was added to cell cultures at 25 ng/mL
and higher (Figures ##FIG##0##1(a)## and ##FIG##0##1(b)##). The 
data in ##FIG##0##Figure 1(c)## also showed the
suppressive effect of EP on VEGF release from mast cells induced by antigenic
stimulation; treatment of mast cells with EP at more than 30 ng/mL significantly
suppressed increase in VEGF levels induced by antigenic stimulation. We then
examined factor levels in water-soluble intracellular extracts by ELISA. As
shown in Figures ##FIG##1##2(a)##, ##FIG##1##2(b)##, and ##FIG##1##2(c)##, 
treatment of mast cells with EP did not
cause the prevention of factor release from cells; the levels of factors (KC,
TNF, and VEGF) in extracts from cells treated with EP at 40 ng/mL are nearly
identical (not significant) to that in stimulated but nontreated control (OVA +
IgE).</p> <title>3.2. Inhibitory effects of EP on the mRNA expression for KC, TNF, and VEGF</title> <p>The second group of experiments were
undertaken to examine the influence of EP on mRNA expression for KC, TNF, and
VEGF after OVA stimulation. Mast cells (5 × 10<sup>5</sup> cells/mL) sensitized
with OVA specific IgE were stimulated with OVA in the presence of 0, 10, 20,
25, 30, and 40 ng/mL EP for 4 hours. mRNA expression was examined by
semiquantitative RT-PCR. As shown in Figures ##FIG##2##3(a)##, ##FIG##2##3(b)##, and ##FIG##2##3(c)##, 
treatment of mast cells with EP suppressed mRNA expression for factors examined, which was
increased by OVA stimulation. The minimum concentration of the agent, which
caused significant suppression, was 25 to 30 ng/mL.</p>
<title>4. DISCUSSION</title> <p>EP is a selective and potent
H<sub>1</sub>-receptor antagonist with no anticholinergic and sedative effect
[##REF##15510239##17##, ##REF##14996515##18##]. Our previous work clearly showed that EP could suppress thymus and
activation-regulated chemokine (TARC) production from human peripheral blood T
cells induced by IL-4 and costimulatory molecule stimulation [##REF##16102259##19##]. It is also observed
that EP could antagonize against IL-4-mediated T cell cytokine imbalance in
vitro [##REF##16534218##20##]. These reports strongly suggest that the modulation of EP on both
cytokine and chemokine
production, which are responsible for the development of allergic immune
responses, consists, in part, of a therapeutic mode of action of the agent on allergic
diseases.</p> <p>The structural changes in the airway walls including
epithelium, submucosa, smooth muscle, and vasculature are referred to as airway
remodeling, which is mainly induced by several types of MMPs, in allergic
diseases such as asthma and allergic rhinitis [##REF##15663564##4##, ##REF##11240267##21##, ##REF##11692106##22##]. Furthermore, there
is accumulated evidence that allergic inflammation causes structural changes in
the nasal wall, which is characterized by angiogenesis and subepithelial
basement membrane hypertrophy [##REF##14656347##2##, ##REF##14713906##3##, ##REF##8556565##23##, ##REF##11069574##24##]. Angiogenesis involves destruction
of the basement membrane by MMPs, migration and proliferation of endothelial
cells, and transformation of endothelial cells to form tubes [##REF##8520966##25##]. This process
is mediated by numerous inducers, including members of fibroblast growth factor
family, VEGF, angiogenin, transforming growth factor, TNF, interleukins and chemokines
[##REF##16159620##7##, ##REF##11359417##26##]. Among them, VEGF is the most potent regulator of angiogenesis and
induces the enhancement of proliferation, migration, and tube formation of
endothelial cells [##REF##16159620##7##, ##REF##11359417##26##]. VEGF promotes secretion of MMP-1 and the expression
of chemokines, as well as intracellular adhesion molecule and E-selectin [##REF##16159620##7##].
VEGF also causes vasodilation, which is responsible for edema, inflammatory cell
infiltration and increase in nasal secretions, through the induction of the endothelial
nitric oxide synthase and the subsequent increase in nitric oxide production
[##REF##16159620##7##, ##REF##11359417##26##]. Furthermore, VEGF stimulates monocyte chemotaxis and contributes to the recruitment of
bone-marrow-derived endothelial cells in angiogenesis [##REF##16159620##7##]. Together with the
present results, showing the suppressive activity of EP on VEGF production may
be interpreted as meaning that some of therapeutic effects of EP in AR depend
on its suppression of VEGF production from mast cells. In addition to VEGF, TNF
is considered to be a multifunctional proinflammatory cytokine that plays a
central role in the initiation and maintenance of many inflammatory diseases,
including asthma and allergic rhinitis. Although TNF is associated predominantly with Th1-dependent inflammation,
recent reports indicate that TNF is essential for the production of the Th2
type cytokines and for infiltration of Th2 T cells into the site of allergic
inflammation [##REF##12847490##27##]. TNF is produced by a variety of inflammatory cells,
especially mast cells and macrophages through IgE-dependent mechanisms and also
enhanced the effect of both IL-4 and IL-10 on antigen-specific IgE production [##REF##12847490##27##, ##REF##17379534##28##]. Furthermore, TNF increased the expression of mRNA for
endothelial-leukocyte adhesion molecule-1 (ELAM-1) and vascular cell adhesion
molecule-1 (VCAM-1), which are essential for eosinophil migration into allergic
inflammatory site, in nasal mucosa after ovalbumin sensitization in mice [##REF##12847490##27##],
suggesting that inhibition of TNF production from mast cells through
IgE-dependent mechanisms may also partially account for the therapeutic
mechanisms of the agent on allergic diseases.</p> <p>KC (murine homolog of IL-8), a member of CXC subfamily,
is produced by several types of immune cells, including epithelial cells,
macrophages, mast cells, and lymphocytes after immunological and nonimmunological
stimulation [##REF##12859561##29##]. Although several reports clearly showed that the presence of
large amount of IL-8, chemoattractants for neutrophils and eosinophils, in
nasal lavage fluid obtained from allergic rhinitis [##REF##9146930##30##–##REF##15051938##32##], the role of
this chemokine is limited in granulocyte infiltration into inflammatory sites
of allergic rhinitis [##REF##12859561##29##]. On the other hand, addition of IL-8 into human
microvascular endothelial cells cultured on the surface of a three-dimensional collagen gel-caused-tube-like
structure formation [##REF##15051938##32##]. It is also reported that administration of anti-IL-8 into rabbit corneas
significantly inhibits newly vessel formation (angiogenesis) induced by implantation with ethylene vinyl acetate pellets containing TNF [##REF##9199336##33##], indicating that IL-8 plays an
essential role in TNF-mediated angiogenesis. From these reports, the inhibitory
action of EP on KC production from mast cells by IgE-dependent mechanisms may
also have important therapeutic implications for allergic diseases.</p> <p>Although the present results clearly show
the inhibitory action of EP on the production of proangiogenic factors from
mast cells by antigenic stimulation through the suppression of mRNA expression,
the precise mechanisms by which EP could inhibit the production of
proangiogenic factors from mast cells are not clear at present. The major stimulus
for mast cell activation is the aggregation of the high affinity receptor for
IgE so-called Fc<italic>ε</italic>RI [##REF##17113919##34##].
Crosslinking of Fc<italic>ε</italic>RI-bound
IgE-molecules with specific allergen activates the receptor-associated tyrosine
kinases and the initiation of further phosphorylation of downstream molecules
such as Syk, phosphatidyl-inositol-3-OH-kinase, and phospholipase C<italic>γ</italic> [##REF##17113919##34##]. These proximal
signaling events activate protein kinase C that ultimately initiates mast cell
effector functions such as degranulation and cytokine production [##REF##17113919##34##]. The
activation of these kinases is also reported to require Ca<sup>2+</sup>, which
is increased by crosslinking of IgE with allergen on mast cell surface [##REF##17135765##35##]. It
has been reported that EP at more than 10<sup>−5</sup> M could inhibit Ca<sup>2+</sup> influx and Ca<sup>2+</sup> release from the intracellular calcium store of mast
cells exposed to compound 48/80 and substance P [##REF##1375955##36##, ##REF##7893266##37##]. Judging from these
reports, it is strongly suggested that EP inhibits the changes in Ca<sup>2+</sup> concentration in cytosole induced by antigenic stimulation and results in
suppression of angiogenic factor production from mast cells. In addition to
these signaling pathways, transcription factor NF-<italic>κ</italic>B
regulates the expression of inflammatory gene products in many different cell
lineages. In mast cells, the expression of TNF, IL-6, and IL-8 gene after Fc<italic>ε</italic>RI ligation
strictly depends on the activation of NF-<italic>κ</italic>B [##REF##17113919##34##, ##REF##17135765##35##]. NF-<italic>κ</italic>B is a
ubiquitous protein transcription factor and normally resides in an inactive
state in cytoplasm. However, when activated it translocates to the nucleus,
binds the DNA, and activates genes. Activation of NF-<italic>κ</italic>B is
induced by degradation and dissociation of I<italic>κ</italic>B, an endogenous inhibitor of NF-<italic>κ</italic>B, through
Ca<sup>2+</sup>-dependent and -independent mechanisms [##REF##16534218##20##]. Together with the
present results, there is also possibility that EP inhibits NF-<italic>κ</italic>B activation
through Ca-independent mechanisms and results in suppression of angiogenic
factor production from mast cells.</p> <p>Pharmacological studies have revealed
that, after oral administration of EP into patients with allergic diseases at a
single dose of 20 mg, plasma concentration of this agent was gradually increased
and attains a plateau at 26.9 ± 9.1 ng/mL [##REF##15606439##38##], suggesting that the findings of
the present in vitro study may reflect the biological function of EP in vivo.</p> <p>In conclusion, the present results demonstrate that the
inhibitory action of EP on angiogenic factor production consists, in part, of a
therapeutic mode of action of the agent on allergic diseases, including atopic
allergy and pollinosis.</p>
<p>Recommended by Steven Kunkel</p> <p>Angiogenesis is an important event both in the development of allergic inflammatory responses and in the pathophysiology of tissue remodeling in allergic diseases. In the present study, therefore, we examined the influence of antihistamines on angiogenesis through the choice of epinastine hydrochloride (EP) and murine mast cells in vitro. Mast cells (5 × 10<sup>5</sup> cells/mL) presensitized with murine IgE specific for ovalbumin (OVA) were stimulated with 10 ng/mL OVA in the presence of various concentrations of EP for 4 hours. The levels of angiogenesis factors, keratinocyte-derived chemokine (KC), tumor necrosis factor-<italic>α</italic> (TNF), and vascular endothelial growth factor (VEGF) in culture supernatants, were examined by ELISA. We also examined mRNA expression for the angiogenesis factors by RT-PCR. EP significantly inhibited the production of KC, TNF, and VEGF induced by IgE-dependent mechanism at more than 25 ng/mL. Semiquantitative analysis using RT-PCR showed that EP also significantly reduced mRNA expressions for KC, TNF, and VEGF. These results strongly suggest that EP suppresses angiogenesis factor production through the inhibition of mRNA expression in mast cells and results in favorable modification of clinical conditions of allergic diseases.</p>
<title>ACKNOWLEDGMENT</title> <p>This study was supported, in part, by Nihon Boehringer Ingelheim Co., Ltd. (Tokyo, Japan).</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Suppressive activity of epinastine hydrochloride (EP) on angiogenic factor production from
mast cells in IgE-dependent mechanism. Murine peritoneal mast cells (5 × 10<sup>5</sup> cells/mL) sensitized with IgE specific for ovalbumin (OVA) were stimulated with
10 ng/mL OVA in the presence of various concentrations of EP for 4 hours. The
levels of angiogenic factors in culture supernatants were analyzed by ELISA.
The data were expressed as the mean pg/mL ± SE of triplicate cultures. KC: keratinocyte-derived chemokine (a); TNF: TNF-<italic>α</italic> (b);
VEGF: vascular endothelial growth factor (c);*<italic>P</italic> &lt; .05 versus control (EP at 0 ng/mL).</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>Influence of epinastine hydrochloride (EP) on angiogenic factor release from mast cells in
IgE-dependent mechanism. Murine peritoneal mast cells (5 × 10<sup>5</sup> cells/mL) sensitized with IgE specific for ovalbumin (OVA) were stimulated with
10 ng/mL OVA. After 4 hours, cells were
collected, sonically disrupted, and the levels of angiogenic factors in
cytosole were examined by ELISA. The data are expressed as the mean pg/mL ± SE
of triplicate cultures. KC: keratinocyte-derived chemokine 
(a); TNF: TNF-<italic>α</italic> (b); VEGF: vascular endothelial growth factor (c);*<italic>P</italic> &lt; .05 
versus control (EP at 0 ng/mL).</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>Suppressive activity of epinastine hydrochrolide (EP) on mRNA expression for angiogenic
factors in mast cells in IgE-mediated mechanism. Murine peritoneal mast cells
(5 × 10<sup>5</sup> cells/mL) sensitized with IgE specific for ovalbumin (OVA)
were stimulated with 10 ng/mL OVA for 4 hours. mRNA expression for
angiogenic factors was examined by
real-time RT-PCR. The data are expressed as the mean ± SE of triplicate
cultures. KC: keratinocyte-derived chemokine (a); TNF: TNF-<italic>α</italic> (b);
VEGF: vascular endothelial growth factor (c);*<italic>P</italic> &lt; .05 versus control (EP at 0 ng/mL).</p></caption></fig>
<graphic xlink:href="MI2008-265095.001"/> <graphic xlink:href="MI2008-265095.002"/> <graphic xlink:href="MI2008-265095.003"/>
[{"label": ["31"], "surname": ["Ciprandi", "Tosca", "Marseglia", "Klersy"], "given-names": ["G", "MA", "GL", "C"], "article-title": ["Relationships between allergic inflammation and nasal airflow in children with seasonal allergic rhinitis"], "italic": ["Annals of Allergy, Asthma and Immunology"], "year": ["2005"], "volume": ["94"], "issue": ["2"], "fpage": ["258"], "lpage": ["261"]}]
Mediators Inflamm. 2008 Aug 19; 2008:265095