Assessment of Lung Eosinophils In Situ Using Immunohistological Staining.

Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.

Assessment of in vivo mast cell reactivity in patients with systemic mastocytosis.

BACKGROUND: Patients with systemic mastocytosis (SM) have clinical signs of mast cell (MC) activation and increased levels of MC mediators. It is unclear whether the increased mediator levels are caused by increased numbers of tissue MCs, or whether these cells in affected individuals have a hyperactive phenotype. OBJECTIVE: To determine reactivity of the skin and the airways to directly acting mediators and indirectly acting mast cell secretagogues in subjects with SM. METHODS: Skin reactivity to morphine and histamine, and airway responsiveness to mannitol and methacholine, was assessed in 15 patients with SM, 11 patients with allergic asthma (A) and 13 healthy controls (HC). Serum tryptase and urinary metabolites of the MC mediators histamine and prostaglandin D2 were measured, as well as ex vivo basophil histamine release. RESULTS: Mast cell mediators in the blood and urine were significantly higher in patients with SM than in HC and A controls. Responsiveness to local activation of skin MCs (by morphine) and airway MCs (by mannitol) was similar in SM and HC groups. Likewise, end-organ responsiveness in the skin to histamine, and in the airways to methacholine, was similar in all three subject groups. There was no evidence of increased basophil reactivity in SM patients. CONCLUSIONS AND CLINICAL RELEVANCE: Mast cells in the skin and airways of subjects with SM do not exhibit hyper-reactivity towards the MC-activating stimuli morphine and mannitol, respectively. Therefore, the highly elevated baseline levels of MC mediators in SM are most likely due to increased MC numbers, rather than altered MC responsiveness. The underlying mechanisms could involve leakage of MC mediators, or dysfunctions in mediator synthesis, storage and release. One clinical implication of our study is that there is no contraindication to perform skin tests using morphine in subjects with mastocytosis.