Lee, N. A.

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.

AbstractBackgroundEosinophils are hallmark cells of allergic Th2 respiratory inflammation. However, the relative importance of eosinophil activation and the induction of effector functions such as the expression ofIL‐13 to allergic Th2 pulmonary disease remain to be defined.MethodsWild‐type or cytokine‐deficient (IL‐13−/−orIL‐4−/−) eosinophils treated with cytokines (GM‐CSF,IL‐4,IL‐33) were adoptively transferred into eosinophil‐deficient recipient mice subjected to allergen provocation using established models of respiratory inflammation. Allergen‐induced pulmonary changes were assessed.ResultsIn contrast to the transfer of untreated blood eosinophils to the lungs of recipient eosinophil deficient mice, which induced no immune/inflammatory changes either in the lung or in the lung draining lymph nodes (LDLN), pretreatment of blood eosinophils withGM‐CSFprior to transfer elicited trafficking of these eosinophils toLDLN. In turn, theseLDLNeosinophils elicited the accumulation of dendritic cells andCD4+T cells to these sameLDLNs without inducing pulmonary inflammation. However, exposure of eosinophils toGM‐CSF,IL‐4, andIL‐33 prior to transfer induced not only immune events in theLDLN, but also allergen‐mediated increases in airway Th2 cytokine/chemokine levels, the subsequent accumulation ofCD4+T cells as well as alternatively activated (M2) macrophages, and the induction of pulmonary histopathologies. Significantly, this allergic respiratory inflammation was dependent on eosinophil‐derivedIL‐13, whereasIL‐4 expression by eosinophils had no significant role.ConclusionThe data demonstrate the differential activation of eosinophils as a function of cytokine exposure and suggest that eosinophil‐specificIL‐13 expression by activated cells is a necessary component of the subsequent allergic Th2 pulmonary pathologies.

SummaryThe role of eosinophils in the progression and resolution of allergic respiratory inflammation is poorly defined despite the commonality of their presence and in some cases their use as a biomarker for disease severity and/or symptom control. However, this ambiguity belies the wealth of insights that have recently been gained through the use of eosinophil‐deficient/attenuated strains of mice that have demonstrated novel immunoregulatory and remodelling/repair functions for these cells in the lung following allergen provocation. Specifically, studies of eosinophil‐deficient mice suggest that eosinophils contribute to events occurring in the lungs following allergen provocation at several key moments: (i) the initiating phase of events leading to Th2‐polarized pulmonary inflammation, (ii) the suppression Th1/Th17 pathways in lung‐draining lymph nodes, (iii) the recruitment of effector Th2 T cells to the lung, and finally, (iv) mechanisms of inflammatory resolution that re‐establish pulmonary homoeostasis. These suggested functions have recently been confirmed and expanded upon using allergen provocation of an inducible eosinophil‐deficient strain of mice (iPHIL) that demonstrated an eosinophil‐dependent mechanism(s) leading to Th2 dominated immune responses in the presence of eosinophils in contrast to neutrophilic as well as mixed Th1/Th17/Th2 variant phenotypes in the absence of eosinophils. These findings highlighted that eosinophils are not exclusively downstream mediators controlled by T cells, dendritic cells (DC) and/or innate lymphocytic cells (ILC2). Instead, eosinophils appear to be more aptly described as significant contributors in complex interrelated pathways that lead to pulmonary inflammation and subsequently promote resolution and the re‐establishment of homoeostatic baseline. In this review, we summarize and put into the context the evolving hypotheses that are now expanding our understanding of the roles eosinophils likely have in the lung following allergen provocation.

AbstractBackgroundSputum eosinophilia has been shown to be a predictor of response to anti‐eosinophil therapies in patients with airway diseases. However, quantitative cell counts and differentials of sputum are labor intensive. The objective of this study was to validate a novel ELISA‐based assay of eosinophil peroxidase (EPX) in sputum as a rapid and reliable marker of airway eosinophils.MethodsThe utility of EPX‐based ELISA as an eosinophil‐specific assay was achieved through comparisons with sputum eosinophil differential counts in freshly prepared and archived patient samples from a variety of clinical settings.ResultsEPX levels in sputum correlated with eosinophil percentage (rs = 0.84) in asthma patients with varying degrees of airway eosinophilia. Significantly, unlike assays of other eosinophil granule proteins (e.g., ECP and EDN), which often detect the presence of these proteins even in asthma patients with neutrophilic bronchitis, EPX‐based ELISA levels are not increased in this subset of asthma patients or in COPD patients lacking evidence of an airway eosinophilia. Moreover, sputum EPX was a surrogate marker of airway eosinophilia in other patient studies (e.g., allergen inhalation and treatment trials the anti‐(IL‐5) therapeutic Mepolizumab™). Finally, EPX levels in cytocentrifuged prepared sputum supernatants correlated with those from rapidly prepared noncentrifuged filtrates of sputum (rs = 0.94).Conclusion and clinical implicationEPX‐based ELISA is a valid, reliable, repeatable, and specific surrogate marker of eosinophils and/or eosinophil degranulation in the sputum of respiratory patients. The novel EPX assay is a valid and reproducible eosinophil‐specific assay that can potentially be developed into a point‐of‐care assessment of eosinophil activity in airway secretions.