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The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclasedependent manner. Furthermore, we identify G protein-coupled receptor 65 (GPR65) as the chief acid-sensing receptor expressed by eosinophils, as GPR65-deficient eosinophils were resistant to acid-induced eosinophil cAMP production and enhanced viability. Notably, GPR65 ؊/؊ mice had attenuated airway eosinophilia and increased apoptosis in 2 distinct models of allergic airway disease. We conclude that eosinophil viability is increased in acidic microenvironments in a cAMP-and GPR65-dependent manner. IntroductionAsthma is a chronic lung disease characterized by episodes of inflammation and narrowing of the airways in response to diverse stimuli. 1 Over the past 2 decades, the incidence of asthma has increased worldwide and is now one of the chief diagnoses responsible for pediatric hospitalization. Extracellular acidosis is commonly observed in inflammatory diseases such as asthma. Several studies have shown that the pH of exhaled breath condensate (EBC) in patients with acute asthma is significantly lower than that of normal subjects. The pH of EBC is mildly alkaline in control persons (7.65 Ϯ 0.20), reaching 5.23 plus or minus 0.21 during asthma exacerbations. 2 Furthermore, the EBC pH of pediatric and adult patients with chronic asthma is also significantly decreased compared with healthy people and correlates with asthma severity. 3,4 Whereas EBC is not a direct measurement of airway lining fluid, airway acidification in asthma is supported by alternative approaches, including direct measurements of bronchial secretions. 2 Although EBC pH is not an asthma-specific biomarker, early clinical studies have suggested that acidification contributes to the disease process as inhalation of buffers that increase airway pH alleviates asthma symptoms. 5 G protein-coupled receptor 65 (GPR65), also known as T-cell death-associated gene 8, belongs to a group of acid-sensing receptors in the G2A G protein-coupled receptor (GPCR) family. [6][7][8] This group includes G2A, GPR65, ovarian cancer GPCR1 (OGR1), and GPR4. Some members of the G2A family, including GPR65, have been shown to be proton sensors in lymphocytes. Acid-sensing capacity is mediated by proton transfer to histidines in the first extracellular loop of GPR65, presumably causing a conformational change in GPR65 that activates G␣ s . This acid-sensing activity has been demonstrated by measuring intracellular cyclic adenosine 5Ј-monophosphate (cAMP) in GPR65-deficient lymphocytes incubated at several proton concentrations. 7 To better understand the functional implications of acidic microenvironments on eosinophils, hallmark cells of asthmatic inflam...

SummaryImmunofluorescence and other methods have been used to probe the self-assembly and internalization of the binary toxin, anthrax lethal toxin (LeTx), in primary murine macrophages. Proteolytic activation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surface. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronaseresistant state, characteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation that PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nicked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowly from the cell surface. These studies show that proteolytic cleavage of PA, in addition to permitting oligomerization and LF binding, also promotes internalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this binary toxin that maximizes self-assembly.

Background: Arginase is significantly upregulated in the lungs in murine models of asthma, as well as in human asthma, but its role in allergic airway inflammation has not been fully elucidated in mice.