Citations

287 Background: Our preliminary immunohistochemical studies of bladder cancer patients have shown that the inflammatory/immune responses associated with bladder cancer appear to be Th2-polarized as judged by the preponderance of tumor infiltrating GATA-3+ (Th2) vs. T-bet+ (Th1) T cells, as well as by evidence of tumor-associated eosinophil infiltration and activation. The standard-of-care treatment for Tis bladder cancer is intravesical administration of BCG, a Th1-polarizing immunomodulator. The aim of the present study was to assess the Th2/Th1 ratio correlation with response to BCG therapy in patients with Tis bladder cancer. Methods: Two groups of patients were studied. Group A included 20 patients that responded to BCG, and Group B included 20 patients that did not respond to therapy. Response to BCG was determined as presence/absence of disease at 6 months post-BCG therapy. The initial diagnostic biopsies of these patients were subjected to immunohistochemical analysis using commercially available antibodies specific for Th2 (GATA-3+) and Th1 (T-bet+) lymphocytes, and our unique monoclonal antibody specific for eosinophil peroxidase (EPX-mAb). Ten random high powered (400X) fields at the maximum focus of mononuclear infiltration were examined and the number of GATA 3+ and T-bet+ tumor infiltrating T cells were counted to define the G/T ratio for each patient sample. Eosinophils and their degranulation activity were counted in the same fields. Results: Patients with response to BCG therapy had a G/T ratio of ≥ 3 and patients with non-response to BCG therapy had a G/T ratio of ≤ 1.5. Correlation of these scores with subsequent patient outcomes following BCG immune therapy demonstrated that patient responsiveness (i.e., tumor elimination at 6 months) trended with the higher G/T ratio patients. In addition, initial biopsies of BCG responsive patients display a higher number of eosinophils and degranulation relative to BCG non-responsive patients. Conclusions: Evaluations of initial biopsies revealed a strong correlation between Th1 and Th2 immune response with BCG therapy outcomes. The development of specific and novel tools to identify/measure Th2 immune responses in Tis bladder cancer provides a unique opportunity to predict outcomes at the time of diagnosis.

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.

Abstract Mouse models of allergen provocation and/or transgenic gene expression have provided significant insights regarding the cellular, molecular, and immune responses linked to the pathologies occurring as a result of allergic respiratory inflammation. Nonetheless, the inability to replicate the eosinophil activities occurring in patients with asthma has limited their usefulness to understand the larger role(s) of eosinophils in disease pathologies. These limitations have led us to develop an allergen-naive double transgenic mouse model that expresses IL-5 systemically from mature T cells and eotaxin-2 locally from lung epithelial cells. We show that these mice develop several pulmonary pathologies representative of severe asthma, including structural remodeling events such as epithelial desquamation and mucus hypersecretion leading to airway obstruction, subepithelial fibrosis, airway smooth muscle hyperplasia, and pathophysiological changes exemplified by exacerbated methacholine-induced airway hyperresponsiveness. More importantly, and similar to human patients, the pulmonary pathologies observed are accompanied by extensive eosinophil degranulation. Genetic ablation of all eosinophils from this double transgenic model abolished the induced pulmonary pathologies, demonstrating that these pathologies are a consequence of one or more eosinophil effector functions.

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.

Abstract Leukotrienes (i.e., products of the 5-lipoxygenase pathway) are thought to be contributors to lung pathologies. Moreover, eosinophils have been linked with pulmonary leukotriene activities both as potential sources of these mediators and as responding effector cells. The objective of the present study was to define the role(s) of leukotrienes in the lung pathologies accompanying eosinophil-associated chronic respiratory inflammation. A transgenic mouse model of chronic T helper (Th) 2–driven inflammation expressing IL-5 from T cells and human eotaxin-2 locally in the lung (I5/hE2) was used to define potential in vivo relationships among eosinophils, leukotrienes, and chronic Th2-polarized pulmonary inflammation. Airway levels of cys-leukotrienes and leukotriene B4 (LTB4) are both significantly elevated in I5/hE2 mice. The eosinophil-mediated airway hyperresponsiveness (AHR) characteristic of these mice was abolished in the absence of leukotrienes (i.e., 5-lipoxygenase–deficient I5/hE2). More importantly, the loss of leukotrienes led to an unexpectedly significant decrease in collagen deposition (i.e., pulmonary fibrosis) that accompanied elevated levels of IL-4/-13 and TGF-β in the lungs of I5/hE2 mice. Further studies using mice deficient for the LTB4 receptor (BLT-1−/− /I5/hE2) and I5/hE2 animals administered a cys-leukotriene receptor antagonist (montelukast) demonstrated that the AHR and the enhanced pulmonary fibrosis characteristic of the I5/hE2 model were uniquely cys-leukotriene–mediated events. These data demonstrate that, similar to allergen challenge models of wild-type mice, cys-leukotrienes underlie AHR in this transgenic model of severe pulmonary Th2 inflammation. These data also suggest that an underappreciated link exists among eosinophils, cys-leukotriene–mediated events, and fibrotic remodeling associated with elevated levels of IL-4/-13 and TGF-β.

Key PointsThe loss of the two most abundant eosinophil granule proteins disrupts the production of blood eosinophils from marrow progenitors. Knockout animals deficient for both MBP-1 and EPX represent a novel strain of mice with a specific and congenital loss of eosinophils.

Abstract The eosinophil-associated ribonuclease (Ear) family in the mouse consists of thirteen genes, eleven of which encode RNases that have physical/functional properties similar to the human Ears, eosinophil-derived neurotoxin and eosinophil cationic protein. The expression of Ear genes in the mouse is confined to sites of known eosinophilopoiesis, with the exception of the lung. Two Ear genes, Ear1 and Ear2, are predominantly expressed in the lungs of naive mice. Total Ear gene expression and RNase activity in bronchoalveolar lavage fluid increases significantly upon the induction of pulmonary inflammation using an ovalbumin (OVA) model of allergic sensitization and challenge. Interestingly, pulmonary Ear11 transcripts, which are absent in naive mice, accumulate as a consequence of OVA-mediated TH2 inflammation in the lung. The induction of Ear11 expression is dependent on the presence of T cells, in particular, CD4+ T lymphocytes. This effect is likely the result of the elaboration of TH2 cytokine levels, because pulmonary instillation of interleukin-4 or interleukin-13 induces the accumulation of Ear11 transcripts in naive animals. This study demonstrates that despite an allergen-mediated pulmonary eosinophilia and earlier studies showing that Ears are constituents of eosinophil secondary granules, alveolar macrophages are a significant source of these RNases in lungs of OVA-treated mice.

ABSTRACTObjective:Differentiation between the common etiologies of dense esophageal eosinophilia such as gastroesophageal reflux disease (GERD) and eosinophilic esophagitis can be difficult. We hypothesized that histologic features may provide diagnostic clues concerning the etiology of esophageal eosinophilia.Methods:We performed a retrospective chart review of 204 children with the diagnosis of esophagitis characterized by ≥15 eosinophils (eos) per high‐power field (HPF) in at least 1 biopsy. We then restricted our analysis to subjects who had received at least 8 weeks of only proton pump inhibitors (PPIs) followed by endoscopy and who had a clinicopathologic response to this treatment. Symptoms, endoscopic findings, and pathologic descriptions were reviewed and an eosinophil peroxidase (EPX) index was determined to assess for degranulation/eosinophil activation.Results:Of the 204 identified charts, 7 subjects identified met the inclusion criteria. Five of these 7 patients showed a clinicopathologic response to PPIs after their follow‐up endoscopy, (mean peak eosinophil count: 92 vs 5 eos/HPF, and EPX index: 39.2 vs 14.6, pre‐ and posttreatment, respectively). Two patients experienced initial resolution of symptoms and esophageal eosinophilia with PPI therapy; however, within 17–23 months they redeveloped symptoms and esophageal eosinophilia while receiving PPI therapy at the time of a third endoscopy (mean peak eosinophil count: 40 vs 11 vs 36 eos/HPF, and EPX index: 44 vs 21 vs 36.5, pre‐, post‐ and posttreatment, respectively). No clinicopathologic features or degranulation patterns differentiated subjects with GERD/PPI responsive esophageal eosinophilia from those who had transient response to PPI treatment.Conclusions:No clinicopathologic features differentiated subjects who responded to PPI treatment. PPI treatment can be helpful to exclude GERD and PPI responsive esophageal eosinophilia but long‐term follow‐up is critical in the management of esophagitis.

Objective Eosinophilic oesophagitis (EoE) is a chronic inflammatory condition of the oesophagus with limited treatment options. No previous transgenic model has specifically targeted the oesophageal mucosa to induce oesophageal eosinophilia. Design We developed a mouse model that closely resembles EoE by utilising oxazolone haptenation in mice with transgenic overexpression of an eosinophil poietic and survival factor (interleukin (IL)-5) in resident squamous oesophageal epithelia. Results Overexpression of IL-5 in the healthy oesophagus was achieved in transgenic mice ( L2-IL5 ) using the squamous epithelial promoter Epstein–Barr virus ED-L2. Oxazolone-challenged L2-IL5 mice developed dose-dependent pan-oesophageal eosinophilia, including eosinophil microabscess formation and degranulation as well as basal cell hyperplasia. Moreover, oesophagi expressed increased IL-13 and the eosinophil agonist chemokine eotaxin-1. Treatment of these mice with corticosteroids significantly reduced eosinophilia and epithelial inflammation. Conclusions L2-IL5 mice provide a novel experimental model that can potentially be used in preclinical testing of EoE-related therapeutics and mechanistic studies identifying pathogenetic features associated with mucosal eosinophilia.