# Influence of Epinastine Hydrochloride, anH1-Receptor Antagonist, on the Function of Mite Allergen-Pulsed Murine Bone Marrow-Derived Dendritic Cells In Vitro and In Vivo

Authors: Ken-Zaburo Oshima; Kazuhito Asano; Ken-Ichi Kanai; Miyuki Suzuki; Harumi Suzaki
Journal: Mediators of Inflammation
 (2009)

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## Abstract

There is established concept that dendritic cells (DCs) play essential roles in the development of allergic immune responses. However, the influence ofH1 receptor antagonists on DC functions is not well defined. The aim of the present study was to examine the effect of epinastine hydrochloride (EP), the most notable histamine H1 receptor antagonists in Japan, on Dermatophagoides farinae (Der f)-pulsed mouse bone marrow-derived DCs in vitro and in vivo. EP at more than 25 ng/mL could significantly inhibit the production of IL-6, TNF-α and IL-10 from Der f-pulsed DCs, which was increased by Der f challenge in vitro. On the other hand, EP increased the ability of Der f-pulsed DCs to produce IL-12. Intranasal instillation of Der f-pulsed DCs resulted in nasal eosinophilia associated with a significant increase in IL-5 levels in nasal lavage fluids. Der f-pulsed and EP-treated DCs significantly inhibited nasal eosinophila and reduced IL-5. These results indicate that EP inhibits the development of Th2 immune responses through the modulation of DC functions and results in favorable modification of clinical status of allergic diseases.


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## Body

## 1. Introduction

Allergic rhinitis is an
inflammation of the nasal passages, usually associated with watery nasal
discharge, sneezing and itching of the nose. These clinical symptoms are also
well accepted to be mediated by vasoactive substances, arachidonic acid
metabolites, cytokines and chemokines secreted from inflammatory cells,
including eosinophils, mast cells, and T cells [1]. The initial response in the
development of allergic inflammation is an allergen, such as mite and pollen,
being presented to the nasal mucosa. This allergen is then recognized as an
antigen by antigen-presenting cells (APCs) and presented to plasma cells. These
cells subsequently produce IgE and attach themselves to mast cells and
eosinophils awaiting re-exposure. Upon re-exposure, an antigen-antibody complex
is formed and activate inflammatory cells to secrete several factors
responsible for allergic immune responses [2].The goal of management of allergic immune
responses, including allergic rhinitis is to reduce the clinical symptoms
caused by the inflammation of affected tissues. Avoidance of the allergens or
minimization of contact with them is the best treatment, but some relief may be
found with the pharmacological medications. Pharmacotherapy available for
allergic rhinitis is vast. Although several studies have demonstrated that
corticosteroids are the most effective agents in the treatment of allergic
airway diseases, including allergic rhinitis, the use of this class of drugs
may be limited because of concerns over unwanted side effects [3]. In contrast,
antihistamines are commonly used as the first-line treatment for allergic
rhinitis and successful results are reported [3–5]. Although primal target of
antihistamines is the histamine H1 receptor, these drugs act as
inhibitors of the synthesis and release of chemical mediators and inflammatory
cytokines from eosinophils and mast cells following immunological and
nonimmunological stimulations [6, 7]. Antihistamines also reported to be able
to suppress the ability of leukocytes to produce inflammatory cytokines and
chemokines in response to inflammatory stimulations [8, 9]. Furthermore, our
previous works clearly showed the suppressive activity of antihistamines,
especially fexofenadine hydrochloride, on nasal fibroblasts to produce
inflammatory mediators, such as nitric oxide and matrix metalloproteinases
induced by inflammatory stimulation [10, 11]. These reports strongly suggest
that antihistamines modulate the function of cells, which are responsible for
the development of allergic inflammation, and results in favorable modification
of the allergic disease state or conditions. On the other hand, there is
established concept that dendritic cells (DCs), the most potent APC in airways,
play essential roles in the development of allergic immune responses through
the production of immunomodulatory cytokines and expression of costimulatory
molecules on their cell surface [12–14]. A number of studies reported the
effects of several types of agents, cysteinyl leukotriens receptor antagonists
[13, 15], β2-adrenargic receptor antagonists [16],
and corticosteroids [13, 16], which are used for the treatment of airway
inflammatory diseases, on DC functions, while a few examined the effects of
antihistamines on DCs.In the present study, we examined the influence of epinastine
hydrochloride (EP), the most notable H1 receptor antagonist in Japan, on the
functions of DCs using mite allergen-pulsed murine bone marrow-derived DCs in vitro and in vivo.


## 2. Materials and Methods

### 2.1. Mice

Specific pathogen-free female BALB/c
mice, 7 weeks old, were purchased from Charles River Japan Inc., (Kanagawa,
Japan) and maintained in our animal facilities at25±2°C, 55±5%
humidity and 12-hour light/dark cycle. Each experimental and control group
consisted of five mice. All animal experimental procedures were approved by the
Animal Care and Use Committee of Showa University.


### 2.2. Reagents

EP was kindly donated from Nippon
Boehringer Ingelheim Co., Ltd. (Tokyo, Japan) as a
preservative-free pure powder. EP was dissolved in RPMI-1640 medium (Sigma
Aldrich, Mo, USA) supplemented with 10% heat-inactivated fetal calf serum
(RPMI-FCS) at a concentration of 1.0 mg/mL, sterilized by passing through a 0.2μm filter and stored as a stock solution at 4°C until used. Mite
extract Dermatophagoides farinae (Der f) was obtained from Cosmo Bio Co.,
Ltd. (Tokyo, Japan). Contamination of
lipopolysaccharide (LPS) in the Der f preparations was <0.96 EU/mg Der f as assessed by Limulus amebocyte lysate test (Sigma Aldrich).
Recombinant mouse granulocyte/macrophage colony stimulating factor (rmGM-CSF),
preservative free, was purchased from R&D Systems Inc. (Minn,
USA).


### 2.3. Induction of DCs

Murine bone marrow-derived DCs were
induced as described previously [17]. Briefly, femurs and tibiae of BALB/c mice
(10 mice) were removed and purified the surrounding muscle tissue. Both ends
were cut and the marrow flushed with phosphate-buffered saline (PBS). After
washing five times with PBS containing 500 U/mL penicillin, 500μg/mL
streptomycin, and 10μg/mL amphotericin B, bone marrow cells were suspended in
RPMI-FCS at a concentration of 2×106cells/mL and cultured in 100 mm culture plates in a final volume of 10.0 mL. At day 3, another 10.0 mL of
RPMI-FCS containing 20.0 ng/mL rmGM-CSF was added to the plates. On day 6, half
of the culture medium was changed with fresh medium containing 5.0 ng/mL
rmGM-CSF.


### 2.4. Preparation of Antigen-Pulsed and EP-Treated DC

DCs were collected on day 8, washed
once with RPMI-FCS, resuspended at2×106cells/mL in RPMI-FCS that
contained 5.0 ng/mL rmGM-CSF. These cells were then introduced into 24-well
culture plates in triplicate that contained various concentrations (0, 20, 25,
30, and 35 ng/mL) of EP in a final volume of 2.0 mL. After 2 hour, Der f was added to cell cultures to give
a final concentration of 100 μg/mL. Cell-free culture supernatants collected
after 24-hour incubation were stored at −80°C
until used. To prepare Der f-pulsed
and EP-treated DCs for in vivo use,
DCs collected on day 8 were cultured with 25.0 ng/mL EP in a final volume of
10.0 mL. After 2 hours, 100 μg/mL Der f was added to cell cultures, and incubated for a further 24 hours. The cells were
then collected, washed three times, and resuspended in PBS at concentration of
2×107cells/mL and used for in vivo experiments.


### 2.5. Immunization with Der F-Pulsed DCs and Exposure to Allergen In Vivo

Naïve BALB/c mice were lightly
anesthetized with ether and divided into six groups (five mice/group). To
prepare non-DC instilled control, mice were received intranasally with 50μg of Der f once daily for 5 consecutive
days in a volume of 50μL. Der f nonpulsed control DCs and Der f-pulsed
DCs (1×106cells/50μL) was applied at the tip of the nose with a
micropipet and inhaled involuntarily [15, 16, 18]. These mice were then
challenged intranasally with either PBS or 50μg of Der f (50μL, each) once daily for 5 consecutive days, which was
started 10 days after cell instillation. The remaining one group of mice were
intranasally instilled with Der f-pulsed
DCs pretreated with 25 ng/mL EP and challenged with Der f in a similar manner.


### 2.6. Preparation of Nasal Cavity Lavage Fluid (NLF)

Mice were killed by intraperitoneal
injection with 1.0 mL of 50 mg/mL sodium pentobarbital (Abbott Laboratories, Ill,  USA) 24 hours after
finalDer f challenge [15, 16], and a
midline incision was performed above the sternum. The trachea was exposed by
blunt dissection and a 28 gauge plastic tube was inserted into trachea above
the carina to nasal cavity. The both sides of nasal cavity were then
simultaneously lavaged with 1.0 mL PBS. Aliquots of the nasal lavage fluid were
then centrifuged at 3000 g at 4°C for 15 minutes, and the supernatants
were collected and stored at −80°C
until use. The pellets spended in 1 mL PBS were used for counting eosinophils
and lymphocytes.


### 2.7. Assay for Cytokines

Concentrations of immunomodulatory
cytokines, IL-12p40 and IL-10, and proinflammatory cytokines, IL-6 and TNF-α
in DCs culture supernatants were measured using commercially available mouse
cytokine ELISA assay kits (R&D Systems Inc.) according to the
manufacturer’s instructions. IL-5 and IFN-γ in NLF were also assayed by mouse
cytokine ELISA assay kits (Pierce Biotechnology Inc., Ill, USA). The detectable
minimum levels of these ELISA kits were 15 pg/mL for IL-12p40, 15.0 pg/mL for
IL-10, 3.0 pg/mL for IL-6, 3.0 pg/mL for TNF-α, 5.0 pg/mL for IL-5, 10.0 pg/mL for
IFN-γ,
respectively.


### 2.8. Counting for Lymphocytes and Eosinophils in NLF

Total number of lymphocytes in NLF
were counted with Turk's solution and hemocytometers. The number of eosinophils
in NLF was examined using Hinkelman’s solution and hemocytometers.


### 2.9. Statistical Analysis

Statistical difference were
examined using analysis of variance (ANOVA), followed by Fisher's PLSD test. AP-value less than .05 denoted the presence of a statistically significant
difference.


## 2.1. Mice

Specific pathogen-free female BALB/c
mice, 7 weeks old, were purchased from Charles River Japan Inc., (Kanagawa,
Japan) and maintained in our animal facilities at25±2°C, 55±5%
humidity and 12-hour light/dark cycle. Each experimental and control group
consisted of five mice. All animal experimental procedures were approved by the
Animal Care and Use Committee of Showa University.


## 2.2. Reagents

EP was kindly donated from Nippon
Boehringer Ingelheim Co., Ltd. (Tokyo, Japan) as a
preservative-free pure powder. EP was dissolved in RPMI-1640 medium (Sigma
Aldrich, Mo, USA) supplemented with 10% heat-inactivated fetal calf serum
(RPMI-FCS) at a concentration of 1.0 mg/mL, sterilized by passing through a 0.2μm filter and stored as a stock solution at 4°C until used. Mite
extract Dermatophagoides farinae (Der f) was obtained from Cosmo Bio Co.,
Ltd. (Tokyo, Japan). Contamination of
lipopolysaccharide (LPS) in the Der f preparations was <0.96 EU/mg Der f as assessed by Limulus amebocyte lysate test (Sigma Aldrich).
Recombinant mouse granulocyte/macrophage colony stimulating factor (rmGM-CSF),
preservative free, was purchased from R&D Systems Inc. (Minn,
USA).


## 2.3. Induction of DCs

Murine bone marrow-derived DCs were
induced as described previously [17]. Briefly, femurs and tibiae of BALB/c mice
(10 mice) were removed and purified the surrounding muscle tissue. Both ends
were cut and the marrow flushed with phosphate-buffered saline (PBS). After
washing five times with PBS containing 500 U/mL penicillin, 500μg/mL
streptomycin, and 10μg/mL amphotericin B, bone marrow cells were suspended in
RPMI-FCS at a concentration of 2×106cells/mL and cultured in 100 mm culture plates in a final volume of 10.0 mL. At day 3, another 10.0 mL of
RPMI-FCS containing 20.0 ng/mL rmGM-CSF was added to the plates. On day 6, half
of the culture medium was changed with fresh medium containing 5.0 ng/mL
rmGM-CSF.


## 2.4. Preparation of Antigen-Pulsed and EP-Treated DC

DCs were collected on day 8, washed
once with RPMI-FCS, resuspended at2×106cells/mL in RPMI-FCS that
contained 5.0 ng/mL rmGM-CSF. These cells were then introduced into 24-well
culture plates in triplicate that contained various concentrations (0, 20, 25,
30, and 35 ng/mL) of EP in a final volume of 2.0 mL. After 2 hour, Der f was added to cell cultures to give
a final concentration of 100 μg/mL. Cell-free culture supernatants collected
after 24-hour incubation were stored at −80°C
until used. To prepare Der f-pulsed
and EP-treated DCs for in vivo use,
DCs collected on day 8 were cultured with 25.0 ng/mL EP in a final volume of
10.0 mL. After 2 hours, 100 μg/mL Der f was added to cell cultures, and incubated for a further 24 hours. The cells were
then collected, washed three times, and resuspended in PBS at concentration of
2×107cells/mL and used for in vivo experiments.


## 2.5. Immunization with Der F-Pulsed DCs and Exposure to Allergen In Vivo

Naïve BALB/c mice were lightly
anesthetized with ether and divided into six groups (five mice/group). To
prepare non-DC instilled control, mice were received intranasally with 50μg of Der f once daily for 5 consecutive
days in a volume of 50μL. Der f nonpulsed control DCs and Der f-pulsed
DCs (1×106cells/50μL) was applied at the tip of the nose with a
micropipet and inhaled involuntarily [15, 16, 18]. These mice were then
challenged intranasally with either PBS or 50μg of Der f (50μL, each) once daily for 5 consecutive days, which was
started 10 days after cell instillation. The remaining one group of mice were
intranasally instilled with Der f-pulsed
DCs pretreated with 25 ng/mL EP and challenged with Der f in a similar manner.


## 2.6. Preparation of Nasal Cavity Lavage Fluid (NLF)

Mice were killed by intraperitoneal
injection with 1.0 mL of 50 mg/mL sodium pentobarbital (Abbott Laboratories, Ill,  USA) 24 hours after
finalDer f challenge [15, 16], and a
midline incision was performed above the sternum. The trachea was exposed by
blunt dissection and a 28 gauge plastic tube was inserted into trachea above
the carina to nasal cavity. The both sides of nasal cavity were then
simultaneously lavaged with 1.0 mL PBS. Aliquots of the nasal lavage fluid were
then centrifuged at 3000 g at 4°C for 15 minutes, and the supernatants
were collected and stored at −80°C
until use. The pellets spended in 1 mL PBS were used for counting eosinophils
and lymphocytes.


## 2.7. Assay for Cytokines

Concentrations of immunomodulatory
cytokines, IL-12p40 and IL-10, and proinflammatory cytokines, IL-6 and TNF-α
in DCs culture supernatants were measured using commercially available mouse
cytokine ELISA assay kits (R&D Systems Inc.) according to the
manufacturer’s instructions. IL-5 and IFN-γ in NLF were also assayed by mouse
cytokine ELISA assay kits (Pierce Biotechnology Inc., Ill, USA). The detectable
minimum levels of these ELISA kits were 15 pg/mL for IL-12p40, 15.0 pg/mL for
IL-10, 3.0 pg/mL for IL-6, 3.0 pg/mL for TNF-α, 5.0 pg/mL for IL-5, 10.0 pg/mL for
IFN-γ,
respectively.


## 2.8. Counting for Lymphocytes and Eosinophils in NLF

Total number of lymphocytes in NLF
were counted with Turk's solution and hemocytometers. The number of eosinophils
in NLF was examined using Hinkelman’s solution and hemocytometers.


## 2.9. Statistical Analysis

Statistical difference were
examined using analysis of variance (ANOVA), followed by Fisher's PLSD test. AP-value less than .05 denoted the presence of a statistically significant
difference.


## 3. Results

### 3.1. Influence of EP on Cytokine Production from DCs in Vitro

The first experiments were
undertaken to examine the influence of EP on cytokine production from DCs afterDer f stimulation in vitro. As shown in Figure 1, DCs could
produce much higher (P<.05) levels of IL-10 in response to Der f stimulation as compared with
nonstimulated DCs. On the other hand, addition of EP into cell cultures dose
dependently suppressed the ability of DCs to produce IL-10 (Figure 1). The
minimum concentration of EP that caused significant suppression was 25 ng/mL
(Figure 1). In contrast to the case of IL-10, IL-12p40 levels in culture
supernatants was further increased by the treatment of DCs with EP, when EP at
more that 25 ng/mL was added to cell cultures (Figure 1). We then examined the
influence of EP on the production of IL-6 and TNF-α
from DCs in response to Der f stimulation. As shown in Figure 2, treatment of DCs with EP at more than 25 ng/mL
significantly suppressed the production of IL-6 and TNF-α,
which were increased by Der f stimulation in vitro.Influence of epinastine
hydrochloride (EP) on immunomodulatory cytokine production from dendritic cells
(DCs) stimulated withDermatophagoides
farinae (Der f) in vitro. DCs derived from bone marrow
of BALB/c mice were stimulated with Der f in the presence of various concentrations of EP for 24 hours. IL-10 and IL12p40
levels in culture supernatants were examined by ELISA. The data are expressed
as the mean pg/mL ± SE of triplicate cultures. This is one of two different
experiments, which gave reproducible results.Influence of epinastine
hydrochloride (EP) on proinflammatory cytokine production from dendritic cells
(DCs) stimulated withDermatophagoides
farinae (Der f) in vitro. DCs derived from bone marrow
of BALB/c mice were stimulated with Der f in the presence of various concentrations of EP for 24 hours. IL-6 and TNF-α
levels in culture supernatants were examined by ELISA. The data are expressed
as the mean pg/mL ± SE of triplicate cultures. This is one of two different
experiments, which gave reproducible results.


### 3.2. Influence of Intranasal Instillation of DCs on the Appearance of Inflammatory Cells in NLF

The second experiments were
designed to examine the influence of intranasal instillation of DCs on
inflammatory cell appearance in nasal wall. As shown in Figure3, intranasal
instillation of Der f, nonpulsed
DCs, nonpulsed DCs and challenged Der f,
and pulsed DCs could not cause the apparent changes in inflammatory cell
appearance in nasal wall: NLF obtained from these groups of mice contained
similar numbers (not significant; P<.05) of both eosinophils and lymphocytes. On the
other hand, NLF obtained from mice instilled with Der f-pulsed, nontreated
DCs and challenged with Der f contained higher numbers (P<.05) of both eosinophils and lymphocytes as
compared with control mice. The data in Figure 3 also clearly indicates that
treatment of Der f-pulsed DCs with EP in vitro prevented significantly (P<.05) increase in eosinophils and lymphocytes in NLF induced by Der f intranasal challenge.Influence of nasal
antigenic challenge on inflammatory cell appearance in nasal lavage fluid
obtained from mice instilled nasally withDermatophagoides
farinae (Der f)-pulsed dendritic
cells. BALB/c mice were instilled nasally with Der f-pulsed and 25 ng/mL epinastine hydrochloride (EP)-treated DCs.
Nasal lavage was performed 24 hours after the final Der f allergen exposure. The data are expressed as the mean number
of cells ± SE of five mice. This is one of two different experiments, which
gave reproducible results. Der f alone: Der f allergen exposure alone; N.DC alone: nonpulsed DC instilled;
N.DC + Der f: nonpulsed DC instilled
and Der f allergen exposure; P. DC
alone: Der f-pulsed DC instilled; P.
DC + Der f: Der f-pulsed DC and Der f allergen exposure; P. DC + Der f +
EP: Der f-pulsed, EP-treated DC and Der f exposure.


### 3.3. Influence of EP on Cytokine Production from DCs In Vivo

The third experiments were carried
out to examine whether in vitro changes in the ability of DCs to produce cytokines was also observed in vivo. NLF obtained from mice
instilled nonpulsed DCs contained much lower levels of IL-5, even when these
mice were challenged intranasally withDer
f (Figure 4). However, intranasal challenge with Der f into mice harboring Der
f-pulsed DCs caused significant increase in IL-5 levels in NLF, and this
activity of Der f-pulsed DCs to
produce IL-5 in vivo was
significantly suppressed by the pretreatment with EP in vitro (Figure 4). We then examined the influence of intranasal
instillation of DCs pretreated with EP on the appearance of IFN-γ
in NLF. As shown in Figure 4, nasal instillation of Der f into mice harbored Der
f-pulsed DCs caused significant increase in IFN-γ
levels in NLF as compared with that from control DCs harboring mice. In vitro treatment of Der f-pulsed DCs with EP could not
suppress the ability of cells to produce IFN-γ in response to intranasal challenge
with Der f. NLF obtained from mice
harboring Der f-pulsed DCs and
treated EP contained similar levels (not significant; P>.05) of IFN-γ
to that from Der f-pulsed and
nontreated DCs.Influence of nasal
antigenic challenge on inflammatory cytokine appearance in nasal lavage fluid
obtained from mice instilled nasally withDermatophagoides
farinae (Der f)-pulsed dendritic
cells. BALB/c mice were instilled nasally with Der f-pulsed and 25 ng/mL epinastine hydrochloride (EP)-treated
DCs. Nasal lavage was performed 24 hours after the final Der f allergen exposure. The data are expressed as the mean pg/mL ±
SE of five mice. This is one of two different experiments, which gave
reproducible results. Der f alone: Der f allergen exposure alone; N. DC
alone: nonpulsed DC instilled; N. DC + Der
f: nonpulsed DC instilled and Der f allergen exposure; P. DC alone: Der f-pulsed
DC instilled; P. DC + Der f: Der f-pulsed DC and Der f allergen exposure; P. DC + Der f + EP: Der f-pulsed,
EP-treated DC and Der f exposure.
N. D.: not detected.


## 3.1. Influence of EP on Cytokine Production from DCs in Vitro

The first experiments were
undertaken to examine the influence of EP on cytokine production from DCs afterDer f stimulation in vitro. As shown in Figure 1, DCs could
produce much higher (P<.05) levels of IL-10 in response to Der f stimulation as compared with
nonstimulated DCs. On the other hand, addition of EP into cell cultures dose
dependently suppressed the ability of DCs to produce IL-10 (Figure 1). The
minimum concentration of EP that caused significant suppression was 25 ng/mL
(Figure 1). In contrast to the case of IL-10, IL-12p40 levels in culture
supernatants was further increased by the treatment of DCs with EP, when EP at
more that 25 ng/mL was added to cell cultures (Figure 1). We then examined the
influence of EP on the production of IL-6 and TNF-α
from DCs in response to Der f stimulation. As shown in Figure 2, treatment of DCs with EP at more than 25 ng/mL
significantly suppressed the production of IL-6 and TNF-α,
which were increased by Der f stimulation in vitro.Influence of epinastine
hydrochloride (EP) on immunomodulatory cytokine production from dendritic cells
(DCs) stimulated withDermatophagoides
farinae (Der f) in vitro. DCs derived from bone marrow
of BALB/c mice were stimulated with Der f in the presence of various concentrations of EP for 24 hours. IL-10 and IL12p40
levels in culture supernatants were examined by ELISA. The data are expressed
as the mean pg/mL ± SE of triplicate cultures. This is one of two different
experiments, which gave reproducible results.Influence of epinastine
hydrochloride (EP) on proinflammatory cytokine production from dendritic cells
(DCs) stimulated withDermatophagoides
farinae (Der f) in vitro. DCs derived from bone marrow
of BALB/c mice were stimulated with Der f in the presence of various concentrations of EP for 24 hours. IL-6 and TNF-α
levels in culture supernatants were examined by ELISA. The data are expressed
as the mean pg/mL ± SE of triplicate cultures. This is one of two different
experiments, which gave reproducible results.


## 3.2. Influence of Intranasal Instillation of DCs on the Appearance of Inflammatory Cells in NLF

The second experiments were
designed to examine the influence of intranasal instillation of DCs on
inflammatory cell appearance in nasal wall. As shown in Figure3, intranasal
instillation of Der f, nonpulsed
DCs, nonpulsed DCs and challenged Der f,
and pulsed DCs could not cause the apparent changes in inflammatory cell
appearance in nasal wall: NLF obtained from these groups of mice contained
similar numbers (not significant; P<.05) of both eosinophils and lymphocytes. On the
other hand, NLF obtained from mice instilled with Der f-pulsed, nontreated
DCs and challenged with Der f contained higher numbers (P<.05) of both eosinophils and lymphocytes as
compared with control mice. The data in Figure 3 also clearly indicates that
treatment of Der f-pulsed DCs with EP in vitro prevented significantly (P<.05) increase in eosinophils and lymphocytes in NLF induced by Der f intranasal challenge.Influence of nasal
antigenic challenge on inflammatory cell appearance in nasal lavage fluid
obtained from mice instilled nasally withDermatophagoides
farinae (Der f)-pulsed dendritic
cells. BALB/c mice were instilled nasally with Der f-pulsed and 25 ng/mL epinastine hydrochloride (EP)-treated DCs.
Nasal lavage was performed 24 hours after the final Der f allergen exposure. The data are expressed as the mean number
of cells ± SE of five mice. This is one of two different experiments, which
gave reproducible results. Der f alone: Der f allergen exposure alone; N.DC alone: nonpulsed DC instilled;
N.DC + Der f: nonpulsed DC instilled
and Der f allergen exposure; P. DC
alone: Der f-pulsed DC instilled; P.
DC + Der f: Der f-pulsed DC and Der f allergen exposure; P. DC + Der f +
EP: Der f-pulsed, EP-treated DC and Der f exposure.


## 3.3. Influence of EP on Cytokine Production from DCs In Vivo

The third experiments were carried
out to examine whether in vitro changes in the ability of DCs to produce cytokines was also observed in vivo. NLF obtained from mice
instilled nonpulsed DCs contained much lower levels of IL-5, even when these
mice were challenged intranasally withDer
f (Figure 4). However, intranasal challenge with Der f into mice harboring Der
f-pulsed DCs caused significant increase in IL-5 levels in NLF, and this
activity of Der f-pulsed DCs to
produce IL-5 in vivo was
significantly suppressed by the pretreatment with EP in vitro (Figure 4). We then examined the influence of intranasal
instillation of DCs pretreated with EP on the appearance of IFN-γ
in NLF. As shown in Figure 4, nasal instillation of Der f into mice harbored Der
f-pulsed DCs caused significant increase in IFN-γ
levels in NLF as compared with that from control DCs harboring mice. In vitro treatment of Der f-pulsed DCs with EP could not
suppress the ability of cells to produce IFN-γ in response to intranasal challenge
with Der f. NLF obtained from mice
harboring Der f-pulsed DCs and
treated EP contained similar levels (not significant; P>.05) of IFN-γ
to that from Der f-pulsed and
nontreated DCs.Influence of nasal
antigenic challenge on inflammatory cytokine appearance in nasal lavage fluid
obtained from mice instilled nasally withDermatophagoides
farinae (Der f)-pulsed dendritic
cells. BALB/c mice were instilled nasally with Der f-pulsed and 25 ng/mL epinastine hydrochloride (EP)-treated
DCs. Nasal lavage was performed 24 hours after the final Der f allergen exposure. The data are expressed as the mean pg/mL ±
SE of five mice. This is one of two different experiments, which gave
reproducible results. Der f alone: Der f allergen exposure alone; N. DC
alone: nonpulsed DC instilled; N. DC + Der
f: nonpulsed DC instilled and Der f allergen exposure; P. DC alone: Der f-pulsed
DC instilled; P. DC + Der f: Der f-pulsed DC and Der f allergen exposure; P. DC + Der f + EP: Der f-pulsed,
EP-treated DC and Der f exposure.
N. D.: not detected.


## 4. Discussion

The present study examined the influence ofDer f and EP on the function of DCs. To
do this, we firstly pulsed DCs with Der f,
and then examined the influence of EP on the production of immunomodulatory
cytokines (IL-10 and IL-12) in vitro.
Second, these DCs were instilled intranasally into naïve mice, followed by Der f exposure, and then cytokine
profile and cellular changes in NLF were compared to examine whether in vitro changes in DCs by EP could be
actually reflect in vivo.DCs are the most potent APCs in priming naïve T and B cells, and they
play a key role in determining the type of immune response [12–14]. Located in
an immature state at the site of antigen entry, DCs actively capture and
process antigens, and migrate to the draining lymph nodes, where they present
processed peptides to naïve T cells and initiate various immune responses [19].
During this process, DCs produce several types of cytokines, which affect the
differentiation of activated T cells into Th1 or Th2 cells [12–14]. The
cytokine IL-12 promotes Th1 responses and stimulates activated T cells to
produce IFN-γ, whereas it inhibits the development of
IL-4-producing Th2 cells in response to D.
pteronyssimus [20]. In addition, IL-12 markedly suppresses the IL-4-induced
IgE production from human peripheral blood leukocytes in vitro [21, 22], suggesting that IL-12 might be capable of
downregulating allergic immune responses. This hypothesis may be supported by
the observation that systemic and intranasal administration of IL-12 into
previously sensitized mice could inhibit the airway hyperreactivity against
specific antigens and metacholine as well as eosinophilia in bronchoalveolar
lavage fluid [23, 24]. It is also supported by the experimental evidence that
mucosal gene transfer of IL-12 via a vaccinia virus vector inhibited the
ability of lung lymphoid cells to produce IL-4 and IL-5, but not IFN-γ
and prevented the development of airway hyperreactivity in a mouse model of
ovalbumin-induced asthma [25]. IL-10 is first identified at the molecular level
as a factor produced by Th2 cells, which inhibited the production of cytokines
by Th1 cells. Although produced by Th2 cells, IL-10 inhibits many Th2 functions
relevant to allergic disorders. IL-10 also inhibits the function of mast cells
and eosinophils, which are associated with allergic responses, and favorably
modulates IgE to IgG4 ratios [26]. On the other hand, IL-10 is reported to
enhance the formation of Th2 cells by downregulating IL-12 production [27],
suggesting that the role of IL-10 on the development of allergic immune
responses is controversial. In general, however, at least at the DC level, it
is likely that DCs induce a Th1 response in high IL-12 and low IL-10, whereas
they induce a Th2 response in low IL-12 and high IL-10 [28, 29]. The present
results clearly showed that EP at 25.0 ng/mL, which is similar to a therapeutic
blood level (26.9±9.1ng/mL) when the patients were orally given EP at 20 mg
[30], exerts both suppressive effect on IL-10 production and enhancive effect
on IL-12 production from DCs. Taken together, therefore, it is reasonably to speculate
that EP inhibits the development of Th2 immune response through the modulation
of the ability of DCs to produce immunomodulatory cytokines, especially IL-10
and IL-12, and results in prevention of propagation of allergic inflammation in
nasal walls. This speculation may be supported by the observation that
administration of soluble ovalbumin together with a monoclonal antibody against
IL-10 receptor to mice led to the enhancement of a Th1 response upon
rechallenge [31].In addition to IL-10 and IL-12, DCs are well known to be able to produce
several types of proinflammatory cytokines. In the present study, we
demonstrated that treatment with EP inhibits the ability of DCs to produce
cytokines such as IL-6 and TNF-α. IL-6 is well known to be a multifunctional
cytokine that can promote the development of inflammatory responses. In
addition to playing essential roles in the process of growth and
differentiation of inflammatory cells such as mast cells and B cells [32], this
cytokine displays other biological properties; IL-6 is the major inducer of
C-reactive protein (CRP) production in the liver, which can enhance the ability
of macrophages and neutrophils to produce inflammatory mediators in local
inflammatory lesions [33, 34]. TNF-α is also well accepted to exert several
specific immunomodulatory effects [35, 36]. Furthermore, these two cytokines
and CRP are reported to be able to enhance the activity of inflammatory cells
to produce free radicals, including nitric oxide and superoxide radicals,
which are the most important final effector molecules of inflammatory diseases
[36]. Together with these reports, the present results may suggest that the
suppressive effect of EP on cytokine production from DCs may underlie the
therapeutic mode of action of EP on allergic diseases such as allergic rhinitis
and atopic dermatitis.Before obtaining the conclusion that the suppressive activity of
antihistamine, especially EP on Dc functions contributes, at least in part, to
the therapeutic mode of action of the agent, it is essential for examining
whether the changes of DC functions in vitro could alter the nasal inflammatory responses against specific
allergic challenge in vivo.
Therefore, we then instilled nasally withDer
f-pulsed DCs treated with EP into naïve mice, and examined the degree of
allergic inflammatory responses in the nasal wall against Der f challenge. The present results of in vivo experiments were consistent with those in vitro experiments and demonstrated that mice instilled with Der f-pulsed DCs developed allergic
inflammation in nasal wall as assessed by eosinophil appearance in NLF, which
concomitant with increased production of IL-5. In contrast, mice instilled with Der f-pulsed DCs treated with EP
showed a reduction in nasal eosinophil count and IL-5 production, but increased
IFN-γ,
indicating that the in vitro functional changes noted in Der f-pulsed
DCs by the treatment with EP in vitro were supported by these in vivo experiments. The mechanisms by which instillation of Der f-pulsed DCs pretreated with EP exerts the suppressive effect
on Th2 type cytokine, IL-5 production in vivo are not clear at present.
Our previous work clearly showed that EP at 25 ng/mL significantly inhibits the
expression of costimulatory molecules, CD40, CD80, and CD86, which are
essential for the development of T cell-dependent immune responses, on human
peripheral blood monocyte-derived DCs (MoDCs) in vitro [37]. It is also reported that EP could decrease the
ability of MoDCs to both stimulate CD4+T cells and phagocyte
antigens in vitro [37], suggesting that
these inhibitory action of EP may be responsible for the modulation of
appearance of T-cell cytokines, IL-5, and IFN-γ in NLF after Der f stimulation.Although the present results strongly indicate that antihistamine,
especially EP, inhibits DCs-induced Th2 skewed immune responses and results in
favorable modification in DCs-induced allergic immune responses in nasal walls,
the mechanism(s) by which EP could modulate DC functions induced by specific
allergen stimulation are not clear at present. Several endogenous mediators
have been reported that modulate DC functions, including adenosine [38],
phosphodiesterase [39], prostaglandins [40], and cAMP [41]. Among these, the
influence of cAMP on cytokine production from DCs was extensively studied and
reported that the increase in cAMP activation caused suppression of IL-12
production and enhanced the ability of cells to produce IL-10 [39]. It is also
reported that rolipram and ciaprost, which are pharmacological agents led to
cAMP activation, enhanced IL-10 production from human peripheral blood monocytes in response
to LPS stimulation [42]. Judging from these reports, the present results may be
interpreted that treatment of DCs with EP as well as azelastine hydrochloride,
an H1 receptor antagonist [43], inhibited cAMP activation, which was
enhanced by Der f stimulation, and
results in changes in the ability of DCs to produce immunomodulatory cytokines.
There is growing evidence that cAMP response element-binding protein (CREB),
which is a 48-κDa  nuclear transcription factor bound to cAMP response element,
modulates the ability of cells to produce proinflammatory cytokines, such as
TNF-α
and IL-6. Inhibition of CREB is reported to decrease IL-6 mRNA expression in
vascular smooth muscle cells and peritoneal mouse macrophages in response to the
stimulation with thrombin [44] and deoxynivalenol [45], respectively. It is also reported that inhibition of
CREB activation suppress the ability of macrophage cell line, RAW264.7, to
produce TNF-α
in response to LPS in vitro [46, 47],
suggesting that EP inhibits IL-6 and TNF-α
production from DCs induced by specific allergen stimulation through the
suppression of CREB activation in vitro.
This speculation may be supported by the observation that terfenadine, an H1 receptor antagonist, exerts the suppressive effect on CREB activation in mouse
cholangiocytes in vitro [48]. Anyway,
further experiments are required to clarify the molecular mechanisms by which
EP could modulate DC functions, especially cytokine production.



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*Source: 738038-2009-04-09.xml*