# Immunohistochemical Localization of the Bradykinin B1 and  B2 Receptors in Human Nasal Mucosa

Authors: Hideaki Shirasaki; Etsuko Kanaizumi; Tetsuo Himi
Journal: Mediators of Inflammation
 (2009)

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## Abstract

Bradykinin (BK) has been tobe thought a potent mediator involved in allergic rhinitis because BK was recovered from the nasal lavage fluid of allergic rhinitis patients after allergen provocation and BK receptor antagonists relief nasal allergic symptoms. Two mammalian BK receptor subtypes, B1 and B2, have been defined based on their pharmacological properties. We investigated the localization of these receptors by immunohistochemistry. Human turbinates were obtained after turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. The immunohistochemical study revealed that epithelial cells, submucosal glands, fibroblast, vascular smooth muscle, vascular endothelial cells, and macrophages showed immunoreactivity for both B1 and B2 receptors. The B2 receptor expression was found in peripheral nerve fibers, whereas the B1 expression was not observed in nerves. The results may have an important clinical implication for understanding the differential roles of BK receptor subtypes on upper airway diseases such as allergic rhinitis and nonallergic rhinitis.


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## Body

## 1. Introduction

The allergic  response is a complex  process involving the interaction of many mediators.
Bradykinin (BK) is a potent inflammatory mediator and its actions are mediated
via specific cell surface receptors which are coupled to G-proteins. Two
mammalian BK receptor subtypes, B1 and B2, have been reported, and the amino
acid sequence of the B1 receptor is 36% identical to the amino acid sequence of
the B2 receptor [1]. Administration of exogenous BK into human nasal airway
causes nasal obstruction, rhinorrhea, and nasal pain [2, 3]. These
effects appear to be mediated by bradykinin B2 receptor because bradykinin B2
receptor antagonists abolish bradykinin-induced nasal obstruction and plasma
extravasation, whereas agonists at the bradykinin B1 receptor do not cause any
symptoms [3]. Icatibant, a bradykinin B2 receptor antagonist, inhibits the
immediate inflammatory response to antigen in subjects with perennial allergic
rhinitis [4, 5]. These reports suggest that BK may
play an important role in the pathogenesis of allergic rhinitis. The previous
autoradiografic study using 125I-BK has demonstrated specific 125I-BK
binding sites mainly exist on the small muscular arteries, venous
sinusoids, and submucosal fibers in human nasal
mucosa [6]. However, there has been no other report about BK receptor expression
in upper airway.In the present study, immunohistochemistry for bradykinin B1 and B2
receptors was
performed to confirm the expression and the distribution of these
receptors in human nasal mucosa.


## 2. Materials and Methods

### 2.1. Tissue Preparation

Human
inferior turbinates were obtained after turbinectomy from 12 patients with nasal
obstruction refractory to medical therapy. Informed consent was obtained from
all patients and this study was approved by the ethics committee of Sapporo
Medical University. All were nonsmokers, and 6
patients had perennial allergy against
mites as defined by questionnaire and CAP test (Pharmacia, Uppsala, Sweden).
All medications, including antibiotics, were prohibited for at least 3 weeks
prior to the study. Demographic and clinical
characteristics of the patients are summarized in Table1. The nasal
mucosal specimens were immediately fixed in 10% formalin for
immunohistochemistry.Table 1
Demographic
characteristics of allergic and nonallergic patients.
Allergic rhinitisNonallergic rhinitisN = 6N = 6Sex (male/female)2/43/3Age31(19–58)39(28–55)Specific IgE to house dust mite (d1) (kU/L)2.7(1.0–13)<0.35Total IgE (kU/L)210(10–387)110(10–185)Blood eosinophils (cells/μL)370(70–690)135(55–240)Current nasal symptoms (number of patients)Nasal obstruction6(all patients)4(all patients)Sneezing40Rhinorrhea32Data expressed as median values and range (in brankets).


### 2.2. Immunohistochemistry

#### 2.2.1. Antibodies

For immunohistochemistry of B1 receptor, rabbit antihuman B1 receptor
polyclonal antibody against a peptide corresponding to C-terminal domain of
human B1 receptor (catalog # LS-A3580, Lifespan Biosciences, Mich USA) was used
at 1:20 dilutions. Similarly, for immunohistochemistry of B2 receptor, rabbit
antihuman B2 receptor polyclonal antibody against a peptide corresponding to
C-terminal domain of human B2 receptor
(catalog # LS-A797, Lifespan Biosciences, Mich, USA) was used at 1:100
dilutions. To identify the subsets of cells expressing each
bradykinin receptor, the following monoclonal antibodies were used: anti-CD68
(KP-1 clone, Dako Corporation, Carpinteria, Calif, USA)
for macrophage, anti-CD31 (JC70A clone, Dako) for vascular endothelial cells,
antihuman fibroblast (5B5 clone, DAKO) for fibroblast, anticytokeratin (AE1/AE3
clone, Dako) for epithelial cells, and antineurofilament protein (2F11 clone,
Dako) for peripheral nerves.


#### 2.2.2. Immunohistochemistry

Deparaffinized sections were initially incubated with 3% H2O2 in methanol for 10 minutes to quench endogenous peroxidase activity. After microwave
treatment (10 minutes at 500 Watt in citrate buffer), the sections were
incubated in blocking solution (10% normal goat serum in PBS) for 30 minutes before
incubation in primary antibody. Then, the sections were incubated with
anti-bradykinin B1 or B2 polyclonal antibody for overnight at 4°C, washed, and
incubated for 30 minutes with EnVision+, Peroxidase (Dako). A further washing
in PBS was followed by developing in DAB (Dako) as a chromogen for signal
visualization. The slides were counterstained Mayer's haematoxylin and
coverslipped using mounting medium.To identify the
subsets of cells expressing each bradykinin receptor,
some sections were stained by immunofluorescence technique. For double
staining, deparaffinized sections were incubated overnight at 4°C with a
combination of rabbit polyclonal antihuman bradykinin B1 or B2  antibody  and one
of mouse monoclonal antihuman phenotypical makers antibody. Sections were washed in PBS and were incubated for 30 minutes
with Alexa Fluor 594-labelled goat antimouse IgG (diluted 1:50; Molecular
Probes, Ore, USA) and Alexa Fluor 488-labelled goat antirabbit IgG (diluted
1:50; Molecular Probes). Sections were mounted with SlowFade antifade
kits (Molecular Probes) and examined under Olympus BX51 microscope, DP70 CCD
camera (Olympus Optical Co., Tokyo, Japan). All images were processed with DP
Controller and DP Manager software (Olympus Optical Co) for image analysis. Using this method, bradykinin B1 or B2 receptor expressing
cells was green, cellular phenotypical makers
were red, and the combined signal is visualized as yellow. Negative controls
were obtained by replacing primary antibodies by mouse IgG1 and rabbit
immunoglobulin fraction (Dako).


## 2.1. Tissue Preparation

Human
inferior turbinates were obtained after turbinectomy from 12 patients with nasal
obstruction refractory to medical therapy. Informed consent was obtained from
all patients and this study was approved by the ethics committee of Sapporo
Medical University. All were nonsmokers, and 6
patients had perennial allergy against
mites as defined by questionnaire and CAP test (Pharmacia, Uppsala, Sweden).
All medications, including antibiotics, were prohibited for at least 3 weeks
prior to the study. Demographic and clinical
characteristics of the patients are summarized in Table1. The nasal
mucosal specimens were immediately fixed in 10% formalin for
immunohistochemistry.Table 1
Demographic
characteristics of allergic and nonallergic patients.
Allergic rhinitisNonallergic rhinitisN = 6N = 6Sex (male/female)2/43/3Age31(19–58)39(28–55)Specific IgE to house dust mite (d1) (kU/L)2.7(1.0–13)<0.35Total IgE (kU/L)210(10–387)110(10–185)Blood eosinophils (cells/μL)370(70–690)135(55–240)Current nasal symptoms (number of patients)Nasal obstruction6(all patients)4(all patients)Sneezing40Rhinorrhea32Data expressed as median values and range (in brankets).


## 2.2. Immunohistochemistry

### 2.2.1. Antibodies

For immunohistochemistry of B1 receptor, rabbit antihuman B1 receptor
polyclonal antibody against a peptide corresponding to C-terminal domain of
human B1 receptor (catalog # LS-A3580, Lifespan Biosciences, Mich USA) was used
at 1:20 dilutions. Similarly, for immunohistochemistry of B2 receptor, rabbit
antihuman B2 receptor polyclonal antibody against a peptide corresponding to
C-terminal domain of human B2 receptor
(catalog # LS-A797, Lifespan Biosciences, Mich, USA) was used at 1:100
dilutions. To identify the subsets of cells expressing each
bradykinin receptor, the following monoclonal antibodies were used: anti-CD68
(KP-1 clone, Dako Corporation, Carpinteria, Calif, USA)
for macrophage, anti-CD31 (JC70A clone, Dako) for vascular endothelial cells,
antihuman fibroblast (5B5 clone, DAKO) for fibroblast, anticytokeratin (AE1/AE3
clone, Dako) for epithelial cells, and antineurofilament protein (2F11 clone,
Dako) for peripheral nerves.


### 2.2.2. Immunohistochemistry

Deparaffinized sections were initially incubated with 3% H2O2 in methanol for 10 minutes to quench endogenous peroxidase activity. After microwave
treatment (10 minutes at 500 Watt in citrate buffer), the sections were
incubated in blocking solution (10% normal goat serum in PBS) for 30 minutes before
incubation in primary antibody. Then, the sections were incubated with
anti-bradykinin B1 or B2 polyclonal antibody for overnight at 4°C, washed, and
incubated for 30 minutes with EnVision+, Peroxidase (Dako). A further washing
in PBS was followed by developing in DAB (Dako) as a chromogen for signal
visualization. The slides were counterstained Mayer's haematoxylin and
coverslipped using mounting medium.To identify the
subsets of cells expressing each bradykinin receptor,
some sections were stained by immunofluorescence technique. For double
staining, deparaffinized sections were incubated overnight at 4°C with a
combination of rabbit polyclonal antihuman bradykinin B1 or B2  antibody  and one
of mouse monoclonal antihuman phenotypical makers antibody. Sections were washed in PBS and were incubated for 30 minutes
with Alexa Fluor 594-labelled goat antimouse IgG (diluted 1:50; Molecular
Probes, Ore, USA) and Alexa Fluor 488-labelled goat antirabbit IgG (diluted
1:50; Molecular Probes). Sections were mounted with SlowFade antifade
kits (Molecular Probes) and examined under Olympus BX51 microscope, DP70 CCD
camera (Olympus Optical Co., Tokyo, Japan). All images were processed with DP
Controller and DP Manager software (Olympus Optical Co) for image analysis. Using this method, bradykinin B1 or B2 receptor expressing
cells was green, cellular phenotypical makers
were red, and the combined signal is visualized as yellow. Negative controls
were obtained by replacing primary antibodies by mouse IgG1 and rabbit
immunoglobulin fraction (Dako).


## 2.2.1. Antibodies

For immunohistochemistry of B1 receptor, rabbit antihuman B1 receptor
polyclonal antibody against a peptide corresponding to C-terminal domain of
human B1 receptor (catalog # LS-A3580, Lifespan Biosciences, Mich USA) was used
at 1:20 dilutions. Similarly, for immunohistochemistry of B2 receptor, rabbit
antihuman B2 receptor polyclonal antibody against a peptide corresponding to
C-terminal domain of human B2 receptor
(catalog # LS-A797, Lifespan Biosciences, Mich, USA) was used at 1:100
dilutions. To identify the subsets of cells expressing each
bradykinin receptor, the following monoclonal antibodies were used: anti-CD68
(KP-1 clone, Dako Corporation, Carpinteria, Calif, USA)
for macrophage, anti-CD31 (JC70A clone, Dako) for vascular endothelial cells,
antihuman fibroblast (5B5 clone, DAKO) for fibroblast, anticytokeratin (AE1/AE3
clone, Dako) for epithelial cells, and antineurofilament protein (2F11 clone,
Dako) for peripheral nerves.


## 2.2.2. Immunohistochemistry

Deparaffinized sections were initially incubated with 3% H2O2 in methanol for 10 minutes to quench endogenous peroxidase activity. After microwave
treatment (10 minutes at 500 Watt in citrate buffer), the sections were
incubated in blocking solution (10% normal goat serum in PBS) for 30 minutes before
incubation in primary antibody. Then, the sections were incubated with
anti-bradykinin B1 or B2 polyclonal antibody for overnight at 4°C, washed, and
incubated for 30 minutes with EnVision+, Peroxidase (Dako). A further washing
in PBS was followed by developing in DAB (Dako) as a chromogen for signal
visualization. The slides were counterstained Mayer's haematoxylin and
coverslipped using mounting medium.To identify the
subsets of cells expressing each bradykinin receptor,
some sections were stained by immunofluorescence technique. For double
staining, deparaffinized sections were incubated overnight at 4°C with a
combination of rabbit polyclonal antihuman bradykinin B1 or B2  antibody  and one
of mouse monoclonal antihuman phenotypical makers antibody. Sections were washed in PBS and were incubated for 30 minutes
with Alexa Fluor 594-labelled goat antimouse IgG (diluted 1:50; Molecular
Probes, Ore, USA) and Alexa Fluor 488-labelled goat antirabbit IgG (diluted
1:50; Molecular Probes). Sections were mounted with SlowFade antifade
kits (Molecular Probes) and examined under Olympus BX51 microscope, DP70 CCD
camera (Olympus Optical Co., Tokyo, Japan). All images were processed with DP
Controller and DP Manager software (Olympus Optical Co) for image analysis. Using this method, bradykinin B1 or B2 receptor expressing
cells was green, cellular phenotypical makers
were red, and the combined signal is visualized as yellow. Negative controls
were obtained by replacing primary antibodies by mouse IgG1 and rabbit
immunoglobulin fraction (Dako).


## 3. Results

As shown in Figure1, the
immunoreactivity for B1 receptor was significantly detected in submucosal
glands, epithelial cells (Figures 1(a), 1(b)), and fibroblasts (Figures 1(c), 1(d)). Immunoreactivity for B2 receptor was significantly detected in
submucosal glands, epithelial cells (Figures 2(a), 2(b)), vascular smooth muscle (Figures 2(c), 2(d)), nerve bundle, and fibroblasts (Figures 2(c), 2(d)). Specificity of the staining was also confirmed by the absence of
labeling with normal rabbit immunoglobulin (Figures 1(e) and
1(f)).Immunohistochemical staining for bradykinin B1
receptor in human allergic (a), (c), (e) and nonallergic (b), (d), (f) nasal mucosa.
Inferior turbinates were stained with antihuman B1 receptor antibody (a)–(d) or normal
rabbit immunoglobulin (e), (f). Immunoreactivity for B1
receptor was significantly detected in submucosal glands (a), (b), epithelial
cells (a), (b), and fibroblasts (c), (d). ep: epithelial
cells; v: blood vessels; g: submucosal glands; n: nerves. Scale bar = 100μm.
(a)(b)(c)(d)(e)(f)Immunohistochemical staining for bradykinin B2 receptor in
human allergic (a), (c), (e) and nonallergic (b), (d), (f) nasal mucosa. Inferior turbinates were stained with
antihuman B2 receptor antibody (a)–(d) or normal rabbit immunoglobulin (e), (f).
Immunoreactivity for B2 receptor was significantly detected in submucosal
glands (), epithelial cells (c), vascular smooth muscle (c), (d), nerve bundle, and
fibroblasts (d). ep: epithelial cells; v: blood vessels; g: submucosal glands;
n: nerves. Scale bar = 100μm.
(a)(b)(c)(d)In order to clarify the cell expressing bradykinin B1 and B2
receptors, we performed double immunofluorescence staining. As shown in Figures3 and 4, epithelial cells, submucosal glands (Figures 3(b) and 4(b)), fibroblast (Figures 3(d) and 4(d)), vascular smooth muscle, and vascular endothelial cells (Figures 3(f) and 4(f)) express both B1 and B2 receptors. The B2 receptor was found in
nerve fibers (Figure 4(h)), whereas the B1 expression was not observed in nerves (Figure 3(h)). As shown in Figure 5, the majority of CD68 positive
macrophages showed immunoreactivity for both B1 and B2 receptors.Identification of subsets of cells expressing the B1 receptor in human allergic nasal mucosa. Single staining immunofluorescence for each cell type (panel (a), (c), (e), and (g) and the dual staining for the cell marker and B1 receptor (panel (b), (d), (f), and (h). The B1 receptor protein (green) shows colocalization with antiphenotypical marker antibody (red) and the combined signal is visualized as yellow. Identification markers for cytokeratin (epithelial cells) (a), (b); fibroblast (c), (d);
CD31 (vascular endothelial cells) (e), (f); neurofilament protein (peripheral nerves) (g), (h). Scale bar = 100μm.
(a)(b)(c)(d)(e)(f)(g)(h)Identification of subsets of cells expressing the B2 receptor in human allergic nasal mucosa. Single staining immunofluorescence for each cell type (panel (a), (c), (e), and (g) and the dual staining for the cell marker and B2 receptor (panel (b), (d), (f), and (h). The B2 receptor protein (green) shows colocalization with antiphenotypical marker antibody (red) and the combined signal is visualized as yellow. Identification markers for cytokeratin (epithelial cells) (a), (b); fibroblast (c), (d);
CD31 (vascular endothelial cells) (e), (f); neurofilament protein (peripheral nerves) (g), (h). Scale bar = 100μm.
(a)(b)(c)(d)(e)(f)(g)(h)Expression of bradykinin B1 (a), (b) and B2 (c), (d)
receptors on macrophages in human allergic nasal mucosa. (a) Macrophages (CD68-positive cells) (red). (b) Overlay image of bradykinin B1 receptor protein (green) and macrophages (CD68-positive) (red). The combined signal is visualized as yellow; (c) macrophages (CD68-positive) (red); (d) Overlay image of bradykinin B2 receptor protein (green); macrophages (CD68-positive cells) (red). Scale bar = 50μm.
(a)(b)(c)(d)The
expression levels of both B1 and B2 receptors on epithelial cells and
fibroblast were higher in allergic nasal mucosa (B1
receptor: Figures1(a) and 1(c); B2 receptor: Figures 2(a) and 2(c)) than nonallergic
nasal mucosa (B1 receptor: Figures 1(b) and 1(d); B2 receptor: Figures 2(b) and 2(d)).The patterns of the other immunohistochemical findings in all 12
cases were remarkably similar, and we could not find any other differences of
B1 and B2 receptors immunoreactivity between on allergic and nonallergic nasal
mucosae. The
summary of the results is shown in Table2.Table 2
Distribution pattern of B1 and B2 receptors in
normal and allergic nasal mucosae.
Normal nasalAllergic nasalmucosamucosaB1B2B1B2Epithelium++++++Submucosal gland++++Nerve−++−++Fibroblast++++++Vascular endothelial cell++++Vascular smooth muscle++++++Inflammatory cell (macrophage)++++++


## 4. Discussion

It is well known that the responses to vasoactive
kinin peptides are mediated through the activation of two receptors termed B1 and B2, which have been defined on the basis
of the structure-function relationships of their agonists and antagonists [7].
The natural agonists of the B2-receptor are the nonapeptide bradykinin (BK) and
the decapeptide Lys-BK (kallidin) which are generated by the proteolytic action
of the serine protease kallikrein from the protein precursor kininogen [8]. BK
and Lys-BK are weak B1 receptor agonist, however, the cleavage of these two
B2-agonists by arginine carboxypeptidases produces the high affinity B1
receptor agonists, [des-Arg9]-BK, and [des-Arg10]-kallidin (DLBK), respectively,
[7]. The B1 receptor is not expressed at significant levels in normal tissues,
but its synthesis can be induced after tissue injury and by inflammatory factor
such as lipopolysaccharide and IL-1 beta [9]. On the other hand, the B2
receptor is constitutively expressed in many
types of the cells including smooth muscle cells, certain neurons, fibroblasts,
and epithelial cells of the lung.In the present study, we confirm the expression of
both bradykinin B1 and B2 receptors in human nasal epithelial cells. BK induced
rise in [Ca2+] in primary cultured human nasal epithelial cells,
suggesting the existence of B2 receptor on human nasal epithelial cells [10]. B1
receptor ligand, Lys-des-Arg-BK activated
extracellular signal-regulated kinase (ERK) and the transcription factor AP-1
in human airway epithelial cell lines A549 and BEAS-2B [11]. Taken together,
these previous observations and our present observations suggest the functional B1 and B2
receptors in human nasal epithelial cells.The present study indicated the significant
expression of B1 and B2 receptor expressions on nasal fibroblasts. It has been
reported that BK stimulated IL-1 [12], IL-6 [13], IL-8 [13, 14], and eotaxin [15]
production in cultured human fibroblasts by increasing its gene expression.
TNF-alpha and IL-1beta both induced an increase in B1 and B2 receptor
expressions in human lung fibroblasts [16]. The observation of local production
of IL-1β during inflammation accompanied by B1-receptor upregulation in
several tissues has resulted in the hypothesis that  this cytokine is directly
involved in B1-receptor upregulation [9].The expression of bradykinin B1
and B2 receptors were found not only on
epithelial cells and fibroblasts, but also  on submucosal glands in the present
study. It has been reported that bradykinin receptors were detected over submucosal gland  in
human and guinea pig airways by in vitro autoradiography [17]. With respect to
the effect of BK on airway submucosal glands, it has been reported that BK
directly stimulates isolated airway submucosal gland cells and induces mucus
glycoprotein and Cl- secretion through the activation of B2 receptor
[18]. Also, BK induces an  increase in short-circuit current across a cultured
gland cell layer from human airways with  [Ca2+] rise, indicating a direct
stimulation of ion transport in airway gland cells by BK [19]. On the other
hand, some investigators have reported that BK had no significant effect on
mucin release from human, feline, or ferret airway explants [6, 20].In contrast to significant both B1
and B2 receptor expressions on epithelial cells, fibroblasts, and submucosal
glands, B2 receptor expression on peripheral nerves, but not B1 receptor
expression, could be detected by immunohistochemistry. B1 receptor was thought
to be generally absent in healthy tissues [21, 22]. In contrast, B2 receptors
are constitutively expressed in a range of cell types including sensory
neurons, and their activation results in excitation and sensitization of
sensory neurons [23, 24]. In B2 receptor agonist, BK can stimulate sensory nerve
ending, causing the release of substance P and other neuropeptides [25]. It has
been shown that the nasal stimulation with BK causes nasal pain [2, 3],
suggesting the existence of the functional B2 receptor on sensory nerves.Using double immunofluorescence
technique, we could confirm the significant expression of both B1 and B2
receptors on macrophages. The potency of kinins to stimulate leukocytes has
been thought dependent on differentiation and especially on the
activation stage of these cells. The differentiation of monocytes into macrophages
is associated with functional and phenotypic changes. It has been shown that
human peripheral monocytes express a low number of kinin B2 binding sites [26].
However, immature, unstimulated human monocytes-derived dendritic cells
constitutively express both B1 and B2 receptors, whereas monocytes did not
express B1 or B2 receptor protein [27], suggesting upregulation of B1 and B2
receptors during the differentiation of the cells. With respect to the effect
of BK on monocytes, BK acting via B2 receptor, increased intracelluar Ca2+, and
stimulated the migration of immature human monocyte-derived dendritic cells
[27]. Thus, it might be possible that local macrophages might be activated by
locally released BK during nasal allergic response.


## 5. Conclusions

Using
immunohistochemical technique, we have demonstrated the distribution of
bradykinin B1 and B2 receptors in human nasal mucosa. Although kinins do not
appear to have major role in allergic rhinitis, our findings should be of
considerable interest for understanding the role of kinins on upper airway
diseases such as allergic rhinitis and nonallergic rhinitis.



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*Source: 102406-2009-04-23.xml*