Anti-Fas mAb-induced apoptosis and cytolysis of airway tissue eosinophils aggravates rather than resolves established inflammation

BACKGROUND: Fas receptor-mediated eosinophil apoptosis is currently forwarded as a mechanism resolving asthma-like inflammation. This view is based on observations in vitro and in airway lumen with unknown translatability to airway tissues in vivo. In fact, apoptotic eosinophils have not been detected in human diseased airway tissues whereas cytolytic eosinophils abound and constitute a major mode of degranulation of these cells. Also, Fas receptor stimulation may bypass the apoptotic pathway and directly evoke cytolysis of non-apoptotic cells. We thus hypothesized that effects of anti-Fas mAb in vivo may include both apoptosis and cytolysis of eosinophils and, hence, that established eosinophilic inflammation may not resolve by this treatment. METHODS: Weeklong daily allergen challenges of sensitized mice were followed by airway administration of anti-Fas mAb. BAL was performed and airway-pulmonary tissues were examined using light and electron microscopy. Lung tissue analysis for CC-chemokines, apoptosis, mucus production and plasma exudation (fibrinogen) were performed. RESULTS: Anti-Fas mAb evoked apoptosis of 28% and cytolysis of 4% of eosinophils present in allergen-challenged airway tissues. Furthermore, a majority of the apoptotic eosinophils remained unengulfed and eventually exhibited secondary necrosis. A striking histopathology far beyond the allergic inflammation developed and included degranulated eosinophils, neutrophilia, epithelial derangement, plasma exudation, mucus-plasma plugs, and inducement of 6 CC-chemokines. In animals without eosinophilia anti-Fas evoked no inflammatory response. CONCLUSION: An efficient inducer of eosinophil apoptosis in airway tissues in vivo, anti-Fas mAb evoked unprecedented asthma-like inflammation in mouse allergic airways. This outcome may partly reflect the ability of anti-Fas to evoke direct cytolysis of non-apoptotic eosinophils in airway tissues. Additionally, since most apoptotic tissue eosinophils progressed into the pro-inflammatory cellular fate of secondary necrosis this may also explain the aggravated inflammation. Our data indicate that Fas receptor mediated eosinophil apoptosis in airway tissues in vivo may cause severe disease exacerbation due to direct cytolysis and secondary necrosis of eosinophils

Analysing the eosinophil cationic protein - a clue to the function of the eosinophil granulocyte

Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil

Rapid and efficient clearance of airway tissue granulocytes through transepithelial migration

Method: Guinea pig tracheobronchial airways where eosinophils are constitutively present in the mucosal tissue were studied. A complex topical stimulus (allergen challenge) was applied and the fate of the eosinophils was determined by selective tracheobronchial lavage and histological examination of the tissue. Results: Within 10 minutes of the allergen challenge, massive migration of eosinophils into the airway lumen occurred together with a reduction in tissue eosinophil numbers. Cell clearance into the lumen continued at high speed and by 30 and 60 minutes the tissue eosinophilia had been reduced by 63% and 73%, respectively. The marked transepithelial migration (estimated maximal speed 35 000 cells/min x cm(2) mucosal surface) took place ubiquitously between epithelial cells without affecting epithelial integrity as assessed by transmission and scanning electron microscopy. Eosinophil apoptosis was not detected but occasional cytolytic eosinophils occurred. Conclusion: This study shows that luminal entry has a remarkably high capacity as a granulocyte elimination process. The data also suggest that an appropriate stimulus of transepithelial migration may be used therapeutically to increase the resolution of inflammatory conditions of airway tissues

A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma

Mouse models of allergic asthma are increasingly used to study the immunopathology of this complex disorder. The degree and type of airway inflammation is often studied by determination of differential cell counts on cytospins of bronchoalveolar lavage fluid (BALF) cells stained with May-Grünwald Giemsa, in which the separation of eosinophils (eos) from neutrophils (neutro) and of monocytes (mono) from activated T cells can be quite problematic. In this study, we compared differential cell counts based on morphological criteria on May-Grünwald Giemsa stained cytospins with a newly developed flow cytometric method. BAL fluid cells were identified based on forward and side scatter characteristics (FSC and SSC), autofluorescence of macrophages, and simultaneous one-step staining with antibodies for T cells (CD3-Cy-Chrome), B cells (B220-Cy-Chrome), eosinophils (CCR3-PE), and dendritic cells (DCs) (MHCII-FITC, CD11c-APC). The validity of this flow cytometric determination was tested by morphological analysis of flow-sorted cellular subsets. In an animal model of ovalbumin-induced asthma, this new method correlated very well with the differential counts based on cytospins. Flow cytometric determination of the cellular composition of BAL fluid in mouse models of asthma is a rapid and easy method that can replace differential cell counts based on morpholog

Eosinophil degranulation in the allergic lung of mice primarily occurs in the airway lumen

Eosinophil degranulation is thought to play a pivotal role in the pathogenesis of allergic disorders. Although mouse models of allergic disorders have been used extensively to identify the contribution of eosinophils to disease, ultrastructural evidence of active granule disassembly has not been reported. In this investigation, we characterized the degree of eosinophil activation in the bone marrow, blood, lung tissue, and airways lumen [bronchoalveolar lavage fluid (BALF)] of ovalbumin-sensitized and aero-challenged wild-type and interleukin-5 transgenic mice. Degranulation was most prominent in and primarily compartmentalized to the airways lumen. Eosinophils released granule proteins by the process of piecemeal degranulation (PMD). Accordingly, recruitment and activation of eosinophils in the lung correlated with the detection of cell-free eosinophil peroxidase in BALF and with the induction of airways hyper-reactivity. As in previous studies with human eosinophils, degranulation of isolated mouse cells did not occur until after adherence to extracellular matrix. However, higher concentrations of exogenous stimuli appear to be required to trigger adherence and degranulation (piecemeal) of mouse eosinophils when compared with values reported for studies of human eosinophils. Thus, mouse eosinophils undergo PMD during allergic inflammation, and in turn, this process may contribute to pathogenesis. However, the degranulation process in the allergic lung of mice is primarily compartmentalized to the airway lumen. Understanding the mechanism of eosinophil degranulation in the airway lumen may provide important insights into how this process occurs in human respiratory diseases.Kristopher Clark, Ljubov Simson, Nicole Newcombe, Aulikki M. L. Koskinen, Joerg Mattes, Nancy A. Lee, James J. Lee, Lindsay A. Dent, Klaus I. Matthaei and Paul S. Foste