Eotaxin-2 regulates newly produced and CD34+ airway eosinophils after allergen exposure

doi:10.1016/j.jaci.2004.03.022

Journal of Allergy and Clinical Immunology

Volume 113, Issue 6, June 2004, Pages 1109-1116

https://doi.org/10.1016/j.jaci.2004.03.022 Get rights and content

Background

Eosinophils develop from CD34⁺ bone marrow progenitors, and evidence increasingly suggests that these progenitors are recruited to the airways in atopic asthmatics after allergen challenge. Moreover, it has been shown that the number of CD34⁺ progenitors in bronchial tissue correlates with asthma severity. To date, however, little is known about how these cells are recruited into the airways.

Objective

Our aim was to evaluate the role of 2 chemokines, eotaxin-1 and/or eotaxin-2, in the recruitment of newly produced and CD34⁺ eosinophils to the airways after allergen exposure.

Methods

BALB/c mice sensitized and exposed to ovalbumin were pretreated intraperitoneally or intranasally with a neutralizing anti-eotaxin-1 and/or anti-eotaxin-2 antibody. A thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU), was used to mark newly produced cells. Bronchoalveolar lavage (BAL), blood, and bone marrow were collected 24 hours after the final exposure.

Results

Anti-eotaxin-1 and/or anti-eotaxin-2 given intranasally (ie, locally to airways) significantly decreased BAL eosinophils. This decrease was paralleled with a decrease in both BrdU⁺ and CD34⁺ eosinophils. In contrast, systemically administered (ie, intraperitoneally) anti-eotaxin-1 and/or anti-eotaxin-2 resulted in a significant decrease in BAL eosinophils only when the combined treatment was of a sufficiently high dosage to produce measurable concentrations in the airways. Furthermore, neither of the treated groups showed any significant decrease in blood or bone marrow eosinophils.

Conclusion

Both eotaxin-1 and eotaxin-2 are involved locally within the airways in the regulation of the recruitment of newly produced and CD34⁺ eosinophils after allergen exposure.

Section snippets

Animals

This study was approved by the Animal Ethics Committee in Gothenburg, Sweden. Male BALB/c mice 5 to 6 weeks old were purchased from Mollegaard-Bommice A/S (Ry, Denmark). The mice were kept under animal housing conditions and provided with food and water ad libitum.

Sensitization and allergen exposure

All mice were immunized twice, at an interval of 5 days by intraperitoneal injections of 0.5 mL alum-precipitated antigen containing 8 μg ovalbumin (Sigma-Aldrich, St Louis, Mo) bound to 4 mg Al(OH)₃ (Sigma-Aldrich) in PBS. Eight days

Concentration of neutralizing antibody in BALF and serum

The intraperitoneal administration of antibody resulted in a high concentration of antibodies in serum and low concentrations in BALF, whereas intranasal administration resulted in a low concentration in serum but high concentrations in BALF (Table I).

Effects of local airway anti-eotaxin-1 treatment

Anti-eotaxin-1 administered intranasally significantly decreased BAL eosinophils compared with isotype controls (54 ± 15 vs 172 ± 18 × 10⁴/mL; P <.01). The inhibiting effect of intranasally administered anti-eotaxin-1 on BAL eosinophils was

Discussion

This study provides evidence that eotaxin-2 is intricately involved in parallel with eotaxin-1 in the eosinophil traffic into the airways after allergen exposure. It is important to note that neutralization of either or both of these chemokines is effective only when treatment results in sufficient concentrations of neutralizing antibodies in BALF. No significant effects were observed in BM or blood after either local airway or systemic administration of the antibodies. In general, the effects

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Thus these data suggest that miR-155 has a significant role in the regulation of airway eosinophilia at the site of inflammation or possible regulation in the recruitment of eosinophils to the airways after allergen challenge. We and others have previously shown that eotaxin-1/CCL11 and eotaxin-2/CCL24 play pivotal roles in regulating eosinophil recruitment locally in the airways in response to allergen challenge.22,23 Thus we measured the levels of these chemokines in BALF from allergen-challenged WT and miR-155 KO mice, respectively.
Allergic asthma is a chronic disease of the conducting airways characterized by T_H2 inflammation and tissue remodeling after exposure to inhaled allergens. Although the T_H2 profile is undisputed, the underlying molecular mechanisms leading to this abnormal T_H2 profile remain largely unclear. MicroRNAs (miRNAs) are short noncoding RNAs that are important regulators of gene expression in the immune system. However, the role of miRNAs, specifically miR-155, in the regulation of allergic airway inflammation is unexplored.
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miR-155 deficiency resulted in diminished eosinophilic inflammation and mucus hypersecretion in the lungs of allergen-sensitized and allergen-challenged mice compared with WT control animals. This was supported by a reduction in T_H2 cell numbers and airway T_H2 cytokine levels and complete abrogation of allergen-induced airway eotaxin-2/CCL24 and periostin levels in miR-155 KO mice. Intranasal instillation of eotaxin-2/CCL24 before allergen challenge partially restored airway eosinophilia in miR-155 KO mice, and adoptive transfer of CD4⁺ T cells resulted in a similar degree of airway eosinophilia in miR-155 KO and WT mice. Furthermore, the transcription factor PU.1, a negative regulator of T_H2 cytokine production, was upregulated in the airways of allergen-challenged miR-155 KO mice compared with WT mice.
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Eosinophil migration into the gut and the release of granular mediators plays a critical role in the pathogenesis of inflammatory bowel diseases, including ulcerative colitis. We recently demonstrated that eosinophil migration into the lung requires cell surface expression of the sialomucin CD34 on mast cells and eosinophils in an asthma model. Based on these findings, we investigated a similar role for CD34 in the migration of eosinophils and other inflammatory cells into the colon as well as explored the effects of CD34 ablation on disease development in a dextran sulfate sodium-induced model of ulcerative colitis. Our findings demonstrate decreased disease severity in dextran sulfate sodium-treated Cd34^−/− mice, as assessed by weight loss, diarrhea, bleeding, colon shortening and tissue pathology, compared with wild-type controls. CD34 was predominantly expressed on eosinophils within inflamed colon tissues, and Cd34^−/− animals exhibited drastically reduced colon eosinophil infiltration. Using chimeric animals, we demonstrated that decreased disease pathology resulted from loss of CD34 from bone marrow-derived cells and that eosinophilia in Cd34^−/−IL5^Tg animals was sufficient to overcome protection from disease. In addition, we demonstrated a decrease in peripheral blood eosinophil numbers following dextran sulfate sodium treatment. These findings demonstrate that CD34 was expressed on colon-infiltrating eosinophils and played a role in eosinophil migration. Further, our findings suggest CD34 is required for efficient eosinophil migration, but not proliferation or expansion, in the development of ulcerative colitis.
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Allergic inflammation is associated with marked infiltration of eosinophils in affected tissues. The eosinophil is believed to be a key effector cells in allergen induced asthma pathogenesis. However, the role of eosinophils in the clinical manifestation of asthma has recently been questioned, since therapies directed against eosinophil infiltration (i.e. anti-interleukin-5) failed to improve clinical symptoms such as airways hyper-responsiveness (AHR) in patients with asthma. Although eosinophils in peripheral blood and the airways were largely depleted after anti-IL-5 treatment, residual eosinophilia in lung tissue persisted, which permits speculation that the remaining eosinophils may be sufficient to drive the asthma symptomatology. Furthermore, recent findings suggest that primitive eosinophil progenitor cells traffic from the bone marrow to sites of inflammation in response to allergen exposure. These progenitors may then differentiate in situ and thus provide an ongoing supply of mature pro-inflammatory cells and secretory mediators that augment the inflammatory response.
In the present article, we will review the evidence for these findings, and discuss the rationale for targeting hematopoiesis and their migration pathways in the treatment of allergic diseases. Furthermore, this review will highlight the hypothesis that both IL-5- and CCR3-mediated signaling pathways may need to be targeted in order to control the inflammation and AHR associated with asthma.
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In 19 patients with a history of aspirin-induced asthma, placebo-controlled bronchial challenges with lysine-aspirin were performed. Peripheral blood was collected before and then 1 hour and 20 hours after challenge (placebo or aspirin). Using the flow-cytometric method, the numbers of leukocyte (CD34⁺ cells) and eosinophil (CD34⁺CD125⁺ cells) progenitors were determined.
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This study demonstrated that bronchial challenge with aspirin may involve systemic reactions and is associated with mobilization of leukocyte and eosinophil progenitor cells from the bone marrow.
CD34 facilitates the development of allergic asthma
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To confirm this observation and to test whether CD34 loss has a role in eosinophil trafficking, we compared BAL eosinophils from wt and CD34−/− mice for expression of CD34 and for their ability to migrate in vitro. To identify eosinophils, BAL fluid and dissociated lung tissue were costained for CD34 and the eotaxin receptor CCR3, a well-known marker of eosinophils that is required for their efficient homing to the lung during allergic inflammation.20 Figure 7A,B shows identical levels of CCR3 expression by wt and CD34−/− BAL eosinophils, suggesting no inherent defect in the ability of these cells to respond to eotaxin.
Asthma is a pulmonary inflammatory disease dependent on eosinophil and mast cell infiltration into the lung. CD34 is a sialomucin expressed by both of these cell types, and we have used CD34^−/− mice and a standard mouse model of asthma to evaluate the importance of CD34 expression on disease development. In comparison with wild-type (wt) mice, CD34^−/− mice exhibited a dramatic reduction in all hallmarks of allergic asthma, including lowered airway inflammatory cell infiltration, airway hyperresponsiveness, and mast-cell recruitment. Bone marrow transplantation experiments confirmed that these defects are due to CD34 expression by bone marrow–derived cells. This was not, however, due to an inability to respond to antigen as, on a per cell basis, wt and CD34^−/− inflammatory cells exhibit identical responses in cytokine production. We found a striking reduction in mobility of CD34^−/− eosinophils in vitro, the major component of inflammatory infiltrates, which was consistent with proposed models for CD34 as an inhibitor of cell-cell adhesion. In summary, our data suggest that CD34 enhances mast-cell and eosinophil invasiveness and that its expression by these cells is a prerequisite for development of allergic asthma.
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: Supported by the Swedish Medical Research Council (K2001-71X-13492-02B), the Swedish Heart Lung Foundation, and the Vårdal Foundation. Prof Jan Lötvall was funded by the Herman Krefting's foundation against asthma/allergy.

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Mechanisms of asthma and allergic inflammationEotaxin-2 regulates newly produced and CD34+ airway eosinophils after allergen exposure

Background

Objective

Methods

Results

Conclusion

Section snippets

Animals

Sensitization and allergen exposure

Concentration of neutralizing antibody in BALF and serum

Effects of local airway anti-eotaxin-1 treatment

Discussion

J Allergy Clin Immunol

J Allergy Clin Immunol

Blood

Immunity

J Allergy Clin Immunol

J Allergy Clin Immunol

Immunol Today

Eosinophilic inflammation in asthma

N Engl J Med

Allergen-induced increases in IL-5 receptor alpha-subunit expression on bone marrow-derived CD34+ cells from asthmatic subjects: a novel marker of progenitor cell commitment towards eosinophilic differentiation

J Clin Invest

Eosinophilopoiesis in a murine model of allergic airway eosinophilia: involvement of bone marrow IL-5 and IL-5 receptor alpha

J Immunol

Mechanisms of acute eosinophil mobilization from the bone marrow stimulated by interleukin-5: the role of specific adhesion molecules and phosphatedylinositol 3-kinase

J Exp Med

Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo

J Exp Med

Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo

J Exp Med

Eotaxin and impaired lung function in asthma

Am J Respir Crit Care Med

Eotaxin-2, a novel CC chemokine that is selective for the chemokine receptor CCR3, and acts like eotaxin on human eosinophil and basophil leucocytes

J Exp Med

Mechanisms of asthma and allergic inflammation
Eotaxin-2 regulates newly produced and CD34⁺ airway eosinophils after allergen exposure