Borchers, Michael T.

AbstractEosinophil migration from circulation is controlled, in part, by chemokines through a family of G-protein-coupled chemokine receptors (CCR). Studies of human eosinophils have demonstrated that signaling through CCR3 receptors is a prominent pathway leading to chemotaxis, although several other receptor-ligand interactions also appear to mediate eosinophil recruitment. The availability of genetically unique strains of mice permits a reductionist approach to assess the signaling pathways in experimental models of human disease. However, despite similarities in these pathways between mice and humans, significant species differences exist, complicating the translation of results from animal models to humans. Purified mouse eosinophils were used in this study to investigate the chemokine receptor expression and the activities of 18 chemokines. Mouse eosinophils isolated from IL-5 transgenic mice expressed transcripts encoding the chemokine receptors CCR1, CCR2, CCR3, CCR5, CCR8, CXCR2, and CXCR4, but not CCR4. Mouse eosinophils also migrated in response to human and mouse eotaxin-1 and -2, but not human eotaxin-3. In addition, the induced migration of mouse eosinophils by TARC, MIP-1β, and KC suggests that unidentified receptor-ligand interactions contribute to eosinophil recruitment. It is interesting that the potent chemoattractant of human eosinophils, RANTES, was unable to mediate mouse eosinophil migration. Furthermore, despite the ability of MIP-1α to bind receptors on purified mouse eosinophils, it was only able to induce significant eosinophil migration in a mixed splenocyte population and was unable to induce migration of highly purified eosinophils. Collectively, these observations reveal physiologically relevant distinctions in mechanisms mediating human and mouse eosinophil migration that potentially reflect evolutionary disparities between these species.

CD4+T cells have a critical role in the development of allergic pulmonary inflammation, including the recruitment of eosinophils to the airway lumen and interstitium. The expression of interleukin (IL)-5 by CD4+cells has, in particular, often been lionized as the central link between allergic inflammation and the concomitant expansion or recruitment of eosinophils. The mechanism(s) by which CD4+T cells mediates eosinophil recruitment was assessed with gene knockout mice deficient for T cells or T cell subtypes and a unique IL-5 transgenic mouse (line NJ.1726) that constitutively overexpresses this cytokine in the lung epithelium. Pulmonary IL-5 expression is significantly attenuated in T cell- and CD4+but not CD8+cell-deficient animals, suggesting an obvious explanation for the lack of eosinophils in the lungs of T cell-deficient and CD4(−/−) mice. However, although the constitutive expression of IL-5 in the lung epithelium of NJ.1726 mice elicited an eosinophilia in the airway lumen of both naive and ovalbumin-treated mice, in the absence of CD4+cells, allergen-mediated eosinophil recruitment to the bronchoalveolar lavage fluid was abolished. Moreover, intranasal instillation of the potent eosinophil-specific chemokine eotaxin-2 was incapable of eliciting eosinophil recruitment in naive and ovalbumin-treated NJ.1726 CD4(−/−) mice, suggesting that eosinophil trafficking during allergic inflammatory responses is a consequence of a CD4+cell-mediated event(s) in addition to IL-5 expression and the establishment of a pulmonary chemokine gradient.

Overexpression of interleukin (IL)-5 by the airway epithelium in mice using the rat CC10 promoter (NJ.1726 line) leads to several histopathologies characteristic of human asthma, including airway hyperreactivity (AHR). We investigated the contribution of B and T cells, as well as CD4 expression, to the development of AHR in IL-5 transgenic mice. NJ.1726 mice on a T cell or CD4 knockout background, but not on a B cell knockout background, lost intrinsic AHR. These effects occurred without decreases in IL-5 or eosinophils. We further investigated the contribution of α4-integrin signaling to the development of AHR in IL-5 transgenic mice through the administration of anti-CD49d (α4-integrin) antibody (PS/2). Administration of PS/2 resulted in immediate (16-h) inhibition of AHR. The inhibition of AHR was not associated with a decrease in airway eosinophils. These studies demonstrate that, despite the presence of increased levels of IL-5 and eosinophils in the lungs of NJ.1726 mice, CD4+cells and α4-integrin signaling are necessary for the intrinsic AHR that develops in IL-5 transgenic mice.

Lymphocyte and/or eosinophil recruitment is dependent on the sequential interactions between adhesion molecules expressed on activated endothelial cells and both leukocyte subtypes. Endothelial P- and E-selectins mediate tethering and rolling of leukocytes through interactions with P-selectin glycoprotein ligand-1 (PSGL-1), and diapedesis subsequently occurs by engagement of endothelial vascular cell adhesion molecule-1 and CD49d (α4-integrins). The anti-inflammatory potential of interfering with these adhesive interactions was assessed with an ovalbumin challenge mouse model of asthma. Administration of a soluble form of PSGL-1 reduced eosinophils (80%) and lymphocytes (50%) in bronchoalveolar lavage fluid without affecting epithelial changes or airway hyperreactivity (AHR). In contrast, although administration of anti-CD49d monoclonal antibodies (PS/2) resulted in similar reductions in eosinophils (75%) and lymphocytes (50%), PS/2 reduced and abolished mucous cell metaplasia and AHR, respectively. Administration of both PSGL-1 and PS/2 had the additive effect of eliminating eosinophils from the airways (96% decrease), with few or no additional reductions (relative to PS/2 administration alone) in lymphocyte recruitment, mucous cell metaplasia, or AHR. These data show that eosinophils and lymphocytes differentially utilize adhesive interactions during recruitment and that the inhibition of AHR is independent of this recruitment.

A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.

The potential role of airway interleukin-5 (IL-5) expression in eliciting mucus production was demonstrated in a pulmonary IL-5 transgenic mouse model (NJ.1726) in which naive transgenic mice display comparable levels of airway mucus relative to allergen-sensitized and -challenged wild-type mice. The intrinsic mucus accumulation of NJ.1726 was abolished in compound transgenic-gene knockout mice deficient of either CD4+cells [NJ.1726/CD4(−/−)] or αβ T cell receptor-positive (TCR+) cells [NJ.1726/αβ TCR(−/−)]. In addition, mucus production in naive NJ.1726 was inhibited by >90% after administration of the soluble anti-IL-4 receptor α-subunit antagonist. The loss of mucus production in NJ.1726/CD4(−/−), NJ.1726/αβ TCR(−/−), and anti-IL-4 receptor α-subunit antagonist-treated mice occurred notwithstanding the significant pulmonary eosinophilia and expansion of airway B cells induced by ectopic IL-5 expression. Furthermore, the loss of mucus accumulation occurred in these mice despite elevated levels of airway and peripheral IL-5, indicating that IL-5 does not directly induce goblet cell metaplasia and mucus production. Thus pulmonary expression of IL-5 alone is capable of inducing CD4+T cell-dependent goblet cell metaplasia, apparently mediated by IL-4 receptor α-subunit-ligand interactions, and represents a previously unrecognized novel pathway for augmenting allergen-induced mucus production.

Allergen-induced recruitment of T lymphocytes and eosinophils to the airways is associated with increased expression of the transcription factor GATA-3. In this study, the relationship between airway inflammation and GATA-3 expression in the lungs was investigated using ragweed-sensitized C57BL/6J mice. Intratracheal ragweed challenge increased both the number of GATA-3-expressing cells in the perivascular and peribronchial regions and the amount of expression per cell. Interleukin (IL)-4 and IL-5 levels in bronchoalveolar lavage fluid were upregulated in parallel with GATA-3 expression. GATA-3 mRNA and protein colocalized to eosinophils. Eosinophils isolated from the lungs and stimulated with phorbol 12-myristate 13-acetate and/or A-23187 released IL-5. The release was inhibited by actinomycin D, which indicates that de novo synthesis of the cytokine was involved. Western blot analysis of proteins from isolated eosinophils demonstrated expression of the p50 subunit of nuclear factor-κB, a transcription factor that is implicated in control of GATA-3 expression. These data provide evidence that allergen challenge increases GATA-3 and proinflammatory cytokine expression by pulmonary eosinophils, which could provide positive feedback for the inflammatory response.

Abstract Allergen provocation of allergic asthma patients is often characterized by an initial period of bronchoconstriction, or early phase reaction (EPR), that leads to maximal airway narrowing within 15–30 min, followed by a recovery period returning airway function to baseline within 1–2 h. In this study, we used a defined OVA provocation model and mice deficient for specific leukocyte populations to investigate the cellular/molecular origins of the EPR. OVA-sensitized/challenged wild-type (C57BL/6J) mice displayed an EPR following OVA provocation. However, this response was absent in gene knockout animals deficient of either B or T cells. Moreover, transfer of OVA-specific IgG, but not IgE, before the OVA provocation, was capable of inducing the EPR in both strains of lymphocyte-deficient mice. Interestingly, an EPR was also observed in sensitized/challenged mast cell-deficient mice following an OVA provocation. These data show that the EPR in the mouse is an immunologically based pathophysiological response that requires allergen-specific IgG but occurs independent of mast cell activities. Thus, in the mouse the initial period of bronchoconstriction following allergen exposure may involve neither mast cells nor IgE-mediated events.

Abstract Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.

Abstract The relationship between eosinophils and the development of Ag-induced pulmonary pathologies, including airway hyper-responsiveness, was investigated using mice deficient for the secondary granule component, major basic protein-1 (mMBP-1). The loss of mMBP-1 had no effect on OVA-induced airway histopathologies or inflammatory cell recruitment. Lung function measurements of knockout mice demonstrated a generalized hyporeactivity to methacholine-induced airflow changes (relative to wild type); however, this baseline phenotype was observable only with methacholine; no relative airflow changes were observed in response to another nonspecific stimulus (serotonin). Moreover, OVA sensitization/aerosol challenge of wild-type and mMBP-1−/− mice resulted in identical dose-response changes to either methacholine or serotonin. Thus, the airway hyper-responsiveness in murine models of asthma occurs in the absence of mMBP-1.