**2. Eosinophils and allergic asthma**

Allergen challenge models have been conceived to reproduce many features of clinical asthma [8]. Actually, atopy, which is the production of allergen-specific IgE antibodies, is a predisposing factor for asthma development, and birth cohort studies have shown that sensitization to allergens such as house dust mite, cat and dog dander and Aspergillus is independent risk factors for wheezing in children [9]. Moreover, exposure to allergens is one of the most recognized environmental factors that trigger asthma symptoms. The term allergic asthma has been used to define the presence of sensitization to environmental allergens and the clinical correlation between exposure and symptoms, both indoor and outdoor allergens being wellknown triggers of asthma exacerbations [10].

Both allergen challenged animal models of asthma and allergic asthma in humans are associated with a T-lymphocyte CD4+ Th2-polarized response as the main feature of airway inflammation. The allergic response is characterized by immediate and late inflammatory responses in which Th2 cells govern the inflammatory cell recruitment and activation by the release of the signature cytokines IL-4, IL-5 and IL-13 as well as IgE antibody synthesis.

### **2.1 Mouse models of allergic asthma**

In acute allergen challenged mouse models of asthma, after the sensitization period (usually 14–21 days), the animal is challenged with the allergen via the airway and this causes many key features of clinical asthma. The analysis of bronchoalveolar lavage (BAL) and bioptic samples of airway walls has supported the hypothesis that asthma is a Th2-mediated disease. A dominating influx of eosinophils has been demonstrated and related to the development of AHR [11]. Moreover, the adoptive transfer of Th2 cells into recipient mice was able to reproduce airway eosinophilia, mucus hypersecretion and AHR after allergen inhalation [12].

However, some of these effects resulted in transient changes and do not involve structural changes. Through chronic allergen exposure in mice, allergen-dependent sensitization, Th2-dependent allergic inflammation, eosinophilic influx into the

**133**

*Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma*

airway mucosa, mucus overproduction and AHR have been reproduced [11, 13]. Generally, acute and chronically treated mice had similar early and late asthmatic responses; however, the acute model had higher levels of eosinophilia, whereas the chronic model showed hyperresponsiveness to lower doses of methacholine and had higher total IgE. On the other hand, many of the lesions observed in chronic human asthma, such as chronic inflammation of the airway wall and airway remodeling

Moreover, transgenic mice that overexpress the Th2 cytokines—IL-4, IL-5, IL-13 and IL-9—in the airway epithelium exhibit the same inflammatory features. IL5 is a Th2 cytokine essential for differentiation, maturation and survival of eosinophils. A key role in allergen-induced inflammatory responses has been shown in murine IL-5-deficient model chronically challenged with an allergen in which the eosinophilia, lung damage and airway hyperreactivity were abolished. The reconstitution of IL-5 production using recombinant vaccinia virus that expressed IL-5 restored eosinophilia and airway dysfunction [14]. Using a clinically relevant model of chronic allergic asthma in mice, Kumar RK et al. showed that anti–IL-5 inhibited inflammation in terms of accumulation of eosinophils in the tracheal epithelium and inflammatory cells in the lamina propria, but had no effect on airway respon-

Many studies have demonstrated the significant role of IL/4IL-13 pathway in asthma. Through the agonization of IL-4R, both IL4 and IL13 activate a tyrosine kinase-dependent signal that after phosphorylation of STAT6 regulates the transcription of Th2-involved genes. Models of IL-4−/− mice were protected from the development of AHR and aspects of remodeling, while the administration of soluble IL-4 receptor reduced inflammation and mucus hypersecretion, but had no effect on AHR [8] Similarly, soluble IL-13 suppressed pulmonary inflammation but

Limitations evidenced in mouse models are that inflammation is not restricted to the conducting airways, but extended to vascular and parenchymal parts of the lung; moreover, some of the clues of asthma inflammation such as the large increases in airway smooth muscle and MC infiltration are not generally observed.

In humans, the role of Th2 cytokines and eosinophils in allergic asthma comes

Sensitizations to environmental allergens in allergic subjects are documented by positive skin prick test reactions and elevated allergen-specific IgE serum levels. Activation of FcεRI on mast cells and basophils by allergen-bound IgE induces the release of preformed vasoactive mediators, which rapidly elicit edema of the bronchial mucosa, mucus production and smooth muscle constriction. This mechanism is confirmed by the increased numbers of cells expressing the high-affinity receptor

Biopsies from bronchial mucosa show CD4+ cell infiltrates and enhanced expression of Th2-type cytokines and chemokines. IL-4 and IL-5 mRNA were localized in activated T cells (CD3+), mast cells (tryptase +) and activated eosinophils (EG2+) both in BAL and bronchial biopsies from mild atopic asthmatic patients [17], and the number of activated CD4+ T cells and IL-5 mRNA positive cells is increased in asthmatic airways following antigen challenge. This skewed cytokine involvement is reflected by the expression of the transcriptional regulators GATA-3 (GATA binding protein 3) after segmental allergen challenge in asthmatics [18]. GATA-3 is a transcriptor factor that finds its binding site in the IL-5 promoter and induces Th2 cytokine gene expression

from many experimental data that in part differ from the mice models.

*DOI: http://dx.doi.org/10.5772/intechopen.92123*

changes, are absent.

siveness to methacholine [15].

had a limited effect on AHR [15].

**2.2 Human models of allergic asthma**

for IgE (FcεRI) in allergic asthmatic tissues [16].

#### *Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma DOI: http://dx.doi.org/10.5772/intechopen.92123*

*Cells of the Immune System*

therapies [6, 7].

**2. Eosinophils and allergic asthma**

known triggers of asthma exacerbations [10].

**2.1 Mouse models of allergic asthma**

IL-4, IL-5 and IL-13 as well as IgE antibody synthesis.

disorder in which many cells and cellular mediators play a role and result in the characteristic pathophysiological changes [2]. The inflammation involves all the airways from the main bronchi to the peripheral small airways. A characteristic pattern of inflammation has been described in asthma involving inflammatory cells mainly mast cells, eosinophils, T lymphocytes, dendritic cells, macrophages and neutrophils, which release mediators that induce symptoms. Both animal models and analysis from human samples have contributed to elucidate the type of inflammation involved in asthma [3]. The most common phenotype of asthma is characterized by eosinophilic airway inflammation and the role of eosinophils as a key player in the pathophysiology of asthma is well documented. Eosinophils emerged as leading cells from the first post-mortem studies of asthmatic lungs, passing through the finding of increased in number and activation status of eosinophils in asthmatic airways [4] and of increased eosinophil surrogates as fractional exhaled nitric oxide (FENO) [5]. Nowadays, the focus is on the definition of the forms of uncontrolled or severe eosinophilic asthma in which airways, sputum and blood eosinophils are consistently increased and represent a biomarker of the eosinophilic endotype of asthma and a guide for biologic target

Allergen challenge models have been conceived to reproduce many features of clinical asthma [8]. Actually, atopy, which is the production of allergen-specific IgE antibodies, is a predisposing factor for asthma development, and birth cohort studies have shown that sensitization to allergens such as house dust mite, cat and dog dander and Aspergillus is independent risk factors for wheezing in children [9]. Moreover, exposure to allergens is one of the most recognized environmental factors that trigger asthma symptoms. The term allergic asthma has been used to define the presence of sensitization to environmental allergens and the clinical correlation between exposure and symptoms, both indoor and outdoor allergens being well-

Both allergen challenged animal models of asthma and allergic asthma in humans are associated with a T-lymphocyte CD4+ Th2-polarized response as the main feature of airway inflammation. The allergic response is characterized by immediate and late inflammatory responses in which Th2 cells govern the inflammatory cell recruitment and activation by the release of the signature cytokines

In acute allergen challenged mouse models of asthma, after the sensitization period (usually 14–21 days), the animal is challenged with the allergen via the airway and this causes many key features of clinical asthma. The analysis of bronchoalveolar lavage (BAL) and bioptic samples of airway walls has supported the hypothesis that asthma is a Th2-mediated disease. A dominating influx of eosinophils has been demonstrated and related to the development of AHR [11]. Moreover, the adoptive transfer of Th2 cells into recipient mice was able to reproduce airway eosinophilia, mucus hypersecretion and AHR after allergen

However, some of these effects resulted in transient changes and do not involve structural changes. Through chronic allergen exposure in mice, allergen-dependent sensitization, Th2-dependent allergic inflammation, eosinophilic influx into the

**132**

inhalation [12].

airway mucosa, mucus overproduction and AHR have been reproduced [11, 13]. Generally, acute and chronically treated mice had similar early and late asthmatic responses; however, the acute model had higher levels of eosinophilia, whereas the chronic model showed hyperresponsiveness to lower doses of methacholine and had higher total IgE. On the other hand, many of the lesions observed in chronic human asthma, such as chronic inflammation of the airway wall and airway remodeling changes, are absent.

Moreover, transgenic mice that overexpress the Th2 cytokines—IL-4, IL-5, IL-13 and IL-9—in the airway epithelium exhibit the same inflammatory features. IL5 is a Th2 cytokine essential for differentiation, maturation and survival of eosinophils. A key role in allergen-induced inflammatory responses has been shown in murine IL-5-deficient model chronically challenged with an allergen in which the eosinophilia, lung damage and airway hyperreactivity were abolished. The reconstitution of IL-5 production using recombinant vaccinia virus that expressed IL-5 restored eosinophilia and airway dysfunction [14]. Using a clinically relevant model of chronic allergic asthma in mice, Kumar RK et al. showed that anti–IL-5 inhibited inflammation in terms of accumulation of eosinophils in the tracheal epithelium and inflammatory cells in the lamina propria, but had no effect on airway responsiveness to methacholine [15].

Many studies have demonstrated the significant role of IL/4IL-13 pathway in asthma. Through the agonization of IL-4R, both IL4 and IL13 activate a tyrosine kinase-dependent signal that after phosphorylation of STAT6 regulates the transcription of Th2-involved genes. Models of IL-4−/− mice were protected from the development of AHR and aspects of remodeling, while the administration of soluble IL-4 receptor reduced inflammation and mucus hypersecretion, but had no effect on AHR [8] Similarly, soluble IL-13 suppressed pulmonary inflammation but had a limited effect on AHR [15].

Limitations evidenced in mouse models are that inflammation is not restricted to the conducting airways, but extended to vascular and parenchymal parts of the lung; moreover, some of the clues of asthma inflammation such as the large increases in airway smooth muscle and MC infiltration are not generally observed.

#### **2.2 Human models of allergic asthma**

In humans, the role of Th2 cytokines and eosinophils in allergic asthma comes from many experimental data that in part differ from the mice models.

Sensitizations to environmental allergens in allergic subjects are documented by positive skin prick test reactions and elevated allergen-specific IgE serum levels. Activation of FcεRI on mast cells and basophils by allergen-bound IgE induces the release of preformed vasoactive mediators, which rapidly elicit edema of the bronchial mucosa, mucus production and smooth muscle constriction. This mechanism is confirmed by the increased numbers of cells expressing the high-affinity receptor for IgE (FcεRI) in allergic asthmatic tissues [16].

Biopsies from bronchial mucosa show CD4+ cell infiltrates and enhanced expression of Th2-type cytokines and chemokines. IL-4 and IL-5 mRNA were localized in activated T cells (CD3+), mast cells (tryptase +) and activated eosinophils (EG2+) both in BAL and bronchial biopsies from mild atopic asthmatic patients [17], and the number of activated CD4+ T cells and IL-5 mRNA positive cells is increased in asthmatic airways following antigen challenge. This skewed cytokine involvement is reflected by the expression of the transcriptional regulators GATA-3 (GATA binding protein 3) after segmental allergen challenge in asthmatics [18]. GATA-3 is a transcriptor factor that finds its binding site in the IL-5 promoter and induces Th2 cytokine gene expression

by biasing Th1/Th2 balance. The increase in GATA-3 expression in the asthmatic subjects correlated significantly with IL-5 expression and AHR [19]. In summary, CD4+ Th2 cells are believed to initiate and perpetuate the inflammatory response in allergic asthma.

IL-5 expression is increased 18–48 h after allergen challenge in BAL samples in mite-associated bronchial asthma when they were stimulated with Dermatophagoides farinae [20]. The levels of IL-5 mRNA-positive cells and IL-5 correlate with the number of eosinophils infiltrating the bronchial mucosa and BAL of asthmatic subjects, with pulmonary function and symptom severity [21]. Biopsies from the respiratory mucosa of allergic asthmatics show the enhanced expression of other Th2-type cytokines and chemokines such as IL-4, IL-6, IL-9, IL-10 and IL-13. Allergen challenge induces in patients with asthma IL13 and IL4 release in BAL and sputum eosinophils that positively correlate with IL-13 expression in asthmatic bronchial submucosa [22]. IL13 is thus involved in the regulation of allergen-induced late-phase inflammatory responses. IL-13, indeed, can modulate the production of IgE through the isotype class switching of B cells; therefore, it is involved in the early phase of allergic reactions.

## **2.3 Recruitment of eosinophils in allergic asthma**

Eosinophils are recruited from progenitors after allergen exposure. Levels of Eo progenitors arise in the peripheral blood after seasonal allergen exposure, during controlled exacerbations of atopic asthma and after single allergen challenge to the airways in atopic asthmatics and animal models. Trafficking of these cells from the bone marrow, where they are produced, to the airways was also demonstrated. In fact, these CD34+ CD45+ progenitors express the IL-5 receptor alpha and are recruited by IL5 and GM-CSF produced in asthmatic airways, subsequently acquiring an activating form that reaches the inflamed airways [23]. Eosinophilopoiesis develops after 24 h from allergen challenges and is followed by the accumulation of eosinophils in the airways.

## **2.4 Eosinophils in different phases of allergic asthma**

The sensitization phase is supposed to be determined by the differentiation of Th naive cells into Th2 lymphocytes. Dendritic cells (DCs) in response to allergen stimulation drive a Th2-oriented response. DC subsets have been described to respond to various stimuli coming from the inflammatory milieu generated after the allergenic encounter. Myeloid CD1c + DCs respond to thymic stromal lymphopoietin (TSLP) produced by the epithelium after allergen encounter by activating allergen-specific memory CD4+ cells [24]. Eosinophils also contribute to the initiation phase of Th2 response by suppressing the Th1/Th17 pathway.

The main role of eosinophils in asthmatic response is yet related to the effector phase of the inflammatory response. After allergen challenge, asthmatics generally develop immediate bronchoconstriction, the so-called early asthmatic response, which is maximized within 30 min and resolves between 1 and 3 h. A proportion of subjects develop a second, delayed bronchoconstrictor response, named the late asthmatic response, which is characterized by prolonged AHR and pronounced airway eosinophilia [25]. So it can be assumed that in isolated early responders a significant or sustained eosinophilic response does not develop. On the other hand, the so-called dual responders develop a sustained IL-5-dependent eosinophilic response in terms of both bone marrow recruitment and sputum accumulation. This response is accompanied by increases in circulating eosinophils, greater

**135**

*Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma*

increases of activated eosinophils in the airways, and the development of airway

Recruited eosinophils in the airways release a variety of toxic products, oxygen radicals, granule-associated cytotoxic proteins and membrane-derived proinflammatory mediators that damage the bronchial epithelium and increase AHR. IL-5 is the most important constituent increasing eosinophil survival, recruitment, degranulation and lung injury following inhalation of antigen, as demonstrated in a segmental antigen lung challenge model [20], and the levels of eosinophils and their cationic proteins in the BAL fluid following allergen challenge correlate with the magnitude of the late phase response. Moreover, a positive correlation between the percentage of BAL eosinophils and the ECP was demonstrated at baseline but not after 4–6 h after allergen inhalation, thus suggesting that eosinophil recruitment and activation seem to follow different temporal kinetics [27]. The effect of IL-5 on eosinophils is demonstrated by the finding of increased expression of the alpha chain of IL-5R mRNA in the bronchial biopsies of atopic and nonatopic asthmatic subjects; the membrane-bound aIL-5R is coexpressed with EG2 in the eosinophils within the bronchial mucosa of asthmatics and inversely

IL-5 acts as chemotactic factors for eosinophils, promoting eosinophil-endothelial adhesion by inducing the expression of VCAM-1 on endothelial cells. In turn, VCAM-1 may bind to integrins on the eosinophils leading to the migration of eosinophils to sites of airway inflammation. Blood eosinophils stimulated with IL-5 adhere to VCAM-1 via the integrins α4β1 and αMβ2 that are the major eosinophil integrin-mediating cell adhesion [29]. Eosinophils obtained from BAL after segmental antigen challenge have both β1 and β2 integrins in a high-activity conformation and adhere to VCAM-1 to a higher degree than blood eosinophils [30]. It seems, therefore, that blood eosinophils are primed by IL-5 or P-selectin (expressed by platelets) to an integrin activation status and are consequently arrested in vessels of inflamed bronchi and move into lung tissue. It is remarkable that the administration of anti–IL-5 can lower β2 integrin activation [31]. IL-5 not only has got the ability to prime eosinophils for subsequent activation but also enhances their survival at sites

The role of other chemokines in allergic asthma is sustained by different pieces of evidence. Eotaxin and regulated on activation, normal T-cell expressed and secreted (RANTES) act on eosinophils inducing chemotaxis as well as specifically activation. In human challenges with the HDM allergen, the peak of eosinophils immunopositive for eotaxin, RANTES and IL-5 occurs at 7 h after allergen inhalation, but persisting eosinophilic airway inflammation and AHR remained for 7 days

These chemokines are released by several cell types in the lung: endothelial cells, epithelial cells, fibroblasts, DCs and smooth muscle cells. Eotaxin creates a chemotactic gradient so that eosinophils pass the endothelium of the blood vessels and migrate to the site of inflammation [33]. Eotaxin has the potential to mobilize eosinophils and their progenitors from bone marrow and this effect is potentiating with that of IL5. Second, in atopic asthmatic patients, high concentrations of eotaxin in BAL fluid are detected as well as an increased expression of eotaxin mRNA and protein in the epithelium and submucosa of their airways. In the airways of allergic asthmatics, eotaxin is in sufficient concentrations to exert chemotactic activity on

eosinophils in vitro and this effect is enhanced by IL-5 [34].

*DOI: http://dx.doi.org/10.5772/intechopen.92123*

hyperresponsiveness [26].

correlated with FEV1 [28].

of allergic inflammation.

after allergen inhalation [32].

**2.5 Eosinophilic chemokines in allergic asthma**

*Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma DOI: http://dx.doi.org/10.5772/intechopen.92123*

*Cells of the Immune System*

response in allergic asthma.

eosinophils in the airways.

is involved in the early phase of allergic reactions.

**2.3 Recruitment of eosinophils in allergic asthma**

**2.4 Eosinophils in different phases of allergic asthma**

tion phase of Th2 response by suppressing the Th1/Th17 pathway.

by biasing Th1/Th2 balance. The increase in GATA-3 expression in the asthmatic subjects correlated significantly with IL-5 expression and AHR [19]. In summary, CD4+ Th2 cells are believed to initiate and perpetuate the inflammatory

Eosinophils are recruited from progenitors after allergen exposure. Levels of Eo progenitors arise in the peripheral blood after seasonal allergen exposure, during controlled exacerbations of atopic asthma and after single allergen challenge to the airways in atopic asthmatics and animal models. Trafficking of these cells from the bone marrow, where they are produced, to the airways was also demonstrated. In fact, these CD34+ CD45+ progenitors express the IL-5 receptor alpha and are recruited by IL5 and GM-CSF produced in asthmatic airways, subsequently acquiring an activating form that reaches the inflamed airways [23]. Eosinophilopoiesis develops after 24 h from allergen challenges and is followed by the accumulation of

The sensitization phase is supposed to be determined by the differentiation of Th naive cells into Th2 lymphocytes. Dendritic cells (DCs) in response to allergen stimulation drive a Th2-oriented response. DC subsets have been described to respond to various stimuli coming from the inflammatory milieu generated after the allergenic encounter. Myeloid CD1c + DCs respond to thymic stromal lymphopoietin (TSLP) produced by the epithelium after allergen encounter by activating allergen-specific memory CD4+ cells [24]. Eosinophils also contribute to the initia-

The main role of eosinophils in asthmatic response is yet related to the effector phase of the inflammatory response. After allergen challenge, asthmatics generally develop immediate bronchoconstriction, the so-called early asthmatic response, which is maximized within 30 min and resolves between 1 and 3 h. A proportion of subjects develop a second, delayed bronchoconstrictor response, named the late asthmatic response, which is characterized by prolonged AHR and pronounced airway eosinophilia [25]. So it can be assumed that in isolated early responders a significant or sustained eosinophilic response does not develop. On the other hand, the so-called dual responders develop a sustained IL-5-dependent eosinophilic response in terms of both bone marrow recruitment and sputum accumulation. This response is accompanied by increases in circulating eosinophils, greater

IL-5 expression is increased 18–48 h after allergen challenge in BAL samples in mite-associated bronchial asthma when they were stimulated with Dermatophagoides farinae [20]. The levels of IL-5 mRNA-positive cells and IL-5 correlate with the number of eosinophils infiltrating the bronchial mucosa and BAL of asthmatic subjects, with pulmonary function and symptom severity [21]. Biopsies from the respiratory mucosa of allergic asthmatics show the enhanced expression of other Th2-type cytokines and chemokines such as IL-4, IL-6, IL-9, IL-10 and IL-13. Allergen challenge induces in patients with asthma IL13 and IL4 release in BAL and sputum eosinophils that positively correlate with IL-13 expression in asthmatic bronchial submucosa [22]. IL13 is thus involved in the regulation of allergen-induced late-phase inflammatory responses. IL-13, indeed, can modulate the production of IgE through the isotype class switching of B cells; therefore, it

**134**

increases of activated eosinophils in the airways, and the development of airway hyperresponsiveness [26].

Recruited eosinophils in the airways release a variety of toxic products, oxygen radicals, granule-associated cytotoxic proteins and membrane-derived proinflammatory mediators that damage the bronchial epithelium and increase AHR.

IL-5 is the most important constituent increasing eosinophil survival, recruitment, degranulation and lung injury following inhalation of antigen, as demonstrated in a segmental antigen lung challenge model [20], and the levels of eosinophils and their cationic proteins in the BAL fluid following allergen challenge correlate with the magnitude of the late phase response. Moreover, a positive correlation between the percentage of BAL eosinophils and the ECP was demonstrated at baseline but not after 4–6 h after allergen inhalation, thus suggesting that eosinophil recruitment and activation seem to follow different temporal kinetics [27].

The effect of IL-5 on eosinophils is demonstrated by the finding of increased expression of the alpha chain of IL-5R mRNA in the bronchial biopsies of atopic and nonatopic asthmatic subjects; the membrane-bound aIL-5R is coexpressed with EG2 in the eosinophils within the bronchial mucosa of asthmatics and inversely correlated with FEV1 [28].

#### **2.5 Eosinophilic chemokines in allergic asthma**

IL-5 acts as chemotactic factors for eosinophils, promoting eosinophil-endothelial adhesion by inducing the expression of VCAM-1 on endothelial cells. In turn, VCAM-1 may bind to integrins on the eosinophils leading to the migration of eosinophils to sites of airway inflammation. Blood eosinophils stimulated with IL-5 adhere to VCAM-1 via the integrins α4β1 and αMβ2 that are the major eosinophil integrin-mediating cell adhesion [29]. Eosinophils obtained from BAL after segmental antigen challenge have both β1 and β2 integrins in a high-activity conformation and adhere to VCAM-1 to a higher degree than blood eosinophils [30]. It seems, therefore, that blood eosinophils are primed by IL-5 or P-selectin (expressed by platelets) to an integrin activation status and are consequently arrested in vessels of inflamed bronchi and move into lung tissue. It is remarkable that the administration of anti–IL-5 can lower β2 integrin activation [31]. IL-5 not only has got the ability to prime eosinophils for subsequent activation but also enhances their survival at sites of allergic inflammation.

The role of other chemokines in allergic asthma is sustained by different pieces of evidence. Eotaxin and regulated on activation, normal T-cell expressed and secreted (RANTES) act on eosinophils inducing chemotaxis as well as specifically activation. In human challenges with the HDM allergen, the peak of eosinophils immunopositive for eotaxin, RANTES and IL-5 occurs at 7 h after allergen inhalation, but persisting eosinophilic airway inflammation and AHR remained for 7 days after allergen inhalation [32].

These chemokines are released by several cell types in the lung: endothelial cells, epithelial cells, fibroblasts, DCs and smooth muscle cells. Eotaxin creates a chemotactic gradient so that eosinophils pass the endothelium of the blood vessels and migrate to the site of inflammation [33]. Eotaxin has the potential to mobilize eosinophils and their progenitors from bone marrow and this effect is potentiating with that of IL5. Second, in atopic asthmatic patients, high concentrations of eotaxin in BAL fluid are detected as well as an increased expression of eotaxin mRNA and protein in the epithelium and submucosa of their airways. In the airways of allergic asthmatics, eotaxin is in sufficient concentrations to exert chemotactic activity on eosinophils in vitro and this effect is enhanced by IL-5 [34].

#### *Cells of the Immune System*

RANTES is also found in high concentrations in the sera in allergic asthma, as well as monocyte chemoattractant protein-1 and -3 (MCP). These chemokines play a role in ongoing lung inflammation, lung leukocyte infiltration, bronchial hyperresponsiveness and the recruitment of eosinophils.

Eotaxins and RANTES bind to the CCR3 receptor expressed on Th2 cells, eosinophils and basophils. Eosinophils in CCR3R knockout mice reach the blood vessels and the endothelium but fail to migrate into lung tissue. Indeed, these mice are protected from AHR after allergen challenges [35]. After antigen challenge, the percentage of CCR3+ eosinophils is downregulated on BAL eosinophils compared with peripheral blood eosinophils, while other chemokine receptors like CCR4, CCR9 and CXCR3 do not, being predominantly involved in activation of eosinophil effector responses [36].

The relationships between the levels of eosinophilic chemokines and AHR or bronchoconstriction are not documented in the same way. Some data suggest that mediators released by cells other than eosinophils, similar to MCs or basophils, can contribute to AHR. In addition, chemokine receptors might be involved in the activation of airway eosinophils for degranulation or prolonged survival. Even if antagonists derived from peptides and small molecules exist to block the chemokine receptor CCR3, the in vivo effect on airway inflammation is not sufficiently proved [33].

Once activated, eosinophils may produce effector molecules like eosinophil major basic protein and eosinophil-derived neurotoxin and degranulate at the site of injury contributing to tissue damage in the asthmatic lung. These molecules have cytotoxic effects on respiratory epithelium, facilitate the entry of other toxic molecules and trigger the degranulation of mast cells and basophils. In asthmatic airways, eosinophils also take part in respiratory-burst–oxidase reactions and generate large amounts of cysteinyl leukotrienes that contribute to increase vascular permeability, mucus secretion and smooth muscle contraction [37].

#### **2.6 Local eosinophilopoiesis**

It has been proposed that CD34+ IL-5Ra+ progenitors after mobilization from the BM during allergen challenge are able to undergo in situ differentiation at the site of allergic inflammation. Actually, CD34+45+IL-5Rα+ progenitors are increased in BAL in mouse models after allergen challenge and precede an increase in BAL eosinophils through a local differentiation via an IL-5-dependent mechanism [38]. Moreover, the CD34+ eosinophil committed pool is maintained within the airways via autocrine IL-5 release and IL-5-induced upregulation of IL-5R. CD34+/IL-5Rα mRNA+ cell number is increased in the airways of asthmatic subjects and related to asthma severity [39]. Surprisingly, eosinophilic precursors persist in the sputum of severe asthmatics that are prednisone resistant after anti-IL-5 treatment [40] and it has been documented that anti-CCR3 strategies do not suppress circulating and airway eosinophils in moderate-to-severe asthmatics. Consequently, it can be hypothesized that blocking local differentiation and expansion of CD34+/IL-5Rα+ cells may reduce eosinophilic inflammation in the airway in asthma.

#### **2.7 Other mechanisms of eosinophil activity into allergic asthmatic airways**

Allergic inflammation is locally perpetuated in the airway by the cross-talk between eosinophils and other resident cells. MCs are activated by MPB and stem cell factor (SCF), both released by eosinophils, contributing, by their direct effects on mast cells, to the perpetuation of allergic inflammation [41].

Eosinophils can also affect fibroblast properties, modulating the process of tissue remodeling. First, eosinophils are the main source in asthma of transforming

**137**

group [48].

*Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma*

growth factor-beta (TGF-β) that induces proliferation and regulates fibroblast function as well as controls the production of proteins of the extracellular matrix (ECM). In turn, tumor necrosis factor-α (TNF-α) derived from mast cells enhances TGF-β synthesis from eosinophils as well as fibroblasts promote survival of MCs and eosinophils by releasing SCF and granulocyte–macrophage-colony stimulating factor (GM-CSF) [42]. Anti-IL-5 humanized monoclonal antibody has been shown to decrease the deposition of many ECM proteins such as collagen III in the RBM of mild atopic asthmatics as well as the number of eosinophils and the degree of

In addition, eosinophils express basic fibroblast growth factor (β-FGF) and VEGF in the submucosa of asthmatic subjects and release many pro-angiogenic

The effect on T-cell immune modulation of eosinophils is more controversial. Cytokine produced by eosinophils may directly influence T-cell selection by DCs determining T-cell tolerance or activation. One example is the induction by IFN-γ of indoleamine 2,3-dioxygenase (IDO) in eosinophils that in turn converts tryptophan (TRYP) to kynurenine (KYN) inducing apoptosis in Th1 cells, while Th2 cells are

The increase of the number of activated Th2 lymphocytes and eosinophils, as well as IL-5 levels, in both BAL fluid and bronchial biopsies from intrinsic asthmatics, has been extensively reported [45]. No difference between atopic and intrinsic asthmatics have been observed in studies examining the expression of high-affinity IgE receptor, IL-5 and IL-4 mRNA and protein expression in bronchial biopsies [16]. Actually, total serum IgE levels have been noted to be increased in the serum of patients with intrinsic asthma. This reflects the increases in Iå and Cå RNA+ cells in the bronchial mucosa and provides evidence for a local IgE synthesis even in the

Eosinophilic infiltration in nonallergic asthma can be even much more than in allergic asthma and this fact is reflected by the finding of a larger amount of RANTES in the bronchoalveolar lavage fluid of patients with nonallergic asthma

Attempts to differentiate the inflammatory cascade between allergic and nonallergic asthma have proposed a different signal in the Th2 pathway of nonallergic asthma attributed to reduced signal transducer and activator of transcription 6 (STAT6) expression and consequently reduced IL-4R signaling in nonallergic asthma [47]. Another peculiar finding was the increased expression of GM-CSF receptor alpha expression in the macrophages detected in mucosa and BAL. Peripheral blood eosinophilia is present both in allergic and nonallergic asthma, in some studies being higher in the former compared to the latter

Different attempts have been found in order to identify an eosinophilic phenotype of asthma. Eosinophilic asthma is reported to account for approximately 50–60% of the total asthma population. The definition of eosinophilic asthma implies that eosinophils are the dominant cells responsible for the pathophysiological changes of the disease. The pathogenic role of eosinophils in these patients is

*DOI: http://dx.doi.org/10.5772/intechopen.92123*

cytokines such as IL-8, IL-6, TGF-β and GM-CSF.

spared from KYN-induced apoptosis by IL-4 [44].

**3. Eosinophils in nonallergic asthma**

absence of a known antigen or allergen trigger.

compared with patients with allergic asthma [46].

**4. The eosinophilic phenotype of asthma**

TGF-α in the BAL fluid [43].

#### *Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma DOI: http://dx.doi.org/10.5772/intechopen.92123*

*Cells of the Immune System*

effector responses [36].

**2.6 Local eosinophilopoiesis**

responsiveness and the recruitment of eosinophils.

RANTES is also found in high concentrations in the sera in allergic asthma, as well as monocyte chemoattractant protein-1 and -3 (MCP). These chemokines play a role in ongoing lung inflammation, lung leukocyte infiltration, bronchial hyper-

Eotaxins and RANTES bind to the CCR3 receptor expressed on Th2 cells, eosinophils and basophils. Eosinophils in CCR3R knockout mice reach the blood vessels and the endothelium but fail to migrate into lung tissue. Indeed, these mice are protected from AHR after allergen challenges [35]. After antigen challenge, the percentage of CCR3+ eosinophils is downregulated on BAL eosinophils compared with peripheral blood eosinophils, while other chemokine receptors like CCR4, CCR9 and CXCR3 do not, being predominantly involved in activation of eosinophil

The relationships between the levels of eosinophilic chemokines and AHR or bronchoconstriction are not documented in the same way. Some data suggest that mediators released by cells other than eosinophils, similar to MCs or basophils, can contribute to AHR. In addition, chemokine receptors might be involved in the activation of airway eosinophils for degranulation or prolonged survival. Even if antagonists derived from peptides and small molecules exist to block the chemokine receptor CCR3, the in vivo effect on airway inflammation is not sufficiently proved [33]. Once activated, eosinophils may produce effector molecules like eosinophil major basic protein and eosinophil-derived neurotoxin and degranulate at the site of injury contributing to tissue damage in the asthmatic lung. These molecules have cytotoxic effects on respiratory epithelium, facilitate the entry of other toxic molecules and trigger the degranulation of mast cells and basophils. In asthmatic airways, eosinophils also take part in respiratory-burst–oxidase reactions and generate large amounts of cysteinyl leukotrienes that contribute to increase vascular

permeability, mucus secretion and smooth muscle contraction [37].

cells may reduce eosinophilic inflammation in the airway in asthma.

on mast cells, to the perpetuation of allergic inflammation [41].

**2.7 Other mechanisms of eosinophil activity into allergic asthmatic airways**

Allergic inflammation is locally perpetuated in the airway by the cross-talk between eosinophils and other resident cells. MCs are activated by MPB and stem cell factor (SCF), both released by eosinophils, contributing, by their direct effects

Eosinophils can also affect fibroblast properties, modulating the process of tissue remodeling. First, eosinophils are the main source in asthma of transforming

It has been proposed that CD34+ IL-5Ra+ progenitors after mobilization from the BM during allergen challenge are able to undergo in situ differentiation at the site of allergic inflammation. Actually, CD34+45+IL-5Rα+ progenitors are increased in BAL in mouse models after allergen challenge and precede an increase in BAL eosinophils through a local differentiation via an IL-5-dependent mechanism [38]. Moreover, the CD34+ eosinophil committed pool is maintained within the airways via autocrine IL-5 release and IL-5-induced upregulation of IL-5R. CD34+/IL-5Rα mRNA+ cell number is increased in the airways of asthmatic subjects and related to asthma severity [39]. Surprisingly, eosinophilic precursors persist in the sputum of severe asthmatics that are prednisone resistant after anti-IL-5 treatment [40] and it has been documented that anti-CCR3 strategies do not suppress circulating and airway eosinophils in moderate-to-severe asthmatics. Consequently, it can be hypothesized that blocking local differentiation and expansion of CD34+/IL-5Rα+

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growth factor-beta (TGF-β) that induces proliferation and regulates fibroblast function as well as controls the production of proteins of the extracellular matrix (ECM). In turn, tumor necrosis factor-α (TNF-α) derived from mast cells enhances TGF-β synthesis from eosinophils as well as fibroblasts promote survival of MCs and eosinophils by releasing SCF and granulocyte–macrophage-colony stimulating factor (GM-CSF) [42]. Anti-IL-5 humanized monoclonal antibody has been shown to decrease the deposition of many ECM proteins such as collagen III in the RBM of mild atopic asthmatics as well as the number of eosinophils and the degree of TGF-α in the BAL fluid [43].

In addition, eosinophils express basic fibroblast growth factor (β-FGF) and VEGF in the submucosa of asthmatic subjects and release many pro-angiogenic cytokines such as IL-8, IL-6, TGF-β and GM-CSF.

The effect on T-cell immune modulation of eosinophils is more controversial. Cytokine produced by eosinophils may directly influence T-cell selection by DCs determining T-cell tolerance or activation. One example is the induction by IFN-γ of indoleamine 2,3-dioxygenase (IDO) in eosinophils that in turn converts tryptophan (TRYP) to kynurenine (KYN) inducing apoptosis in Th1 cells, while Th2 cells are spared from KYN-induced apoptosis by IL-4 [44].
