Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis.

BACKGROUND: The human eosinophil Charcot-Leyden crystal (CLC) protein is a member of the Galectin superfamily and is also known as galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals that are considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. OBJECTIVE: We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. METHODS: Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by coimmunoprecipitation and coaffinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by using enzyme activity assays, confocal microscopy, and short hairpin RNA knockout of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils. RESULTS: CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases (RNases), namely, eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3), and with murine eosinophil-associated RNases. The interaction is independent of glycosylation and is not inhibitory toward endoRNase activity. Activation of eosinophils with INF-γ induces the rapid colocalization of CLC/Gal-10 with eosinophil-derived neurotoxin/RNS2 and CD63. Short hairpin RNA knockdown of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis. CONCLUSIONS: CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic RNases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.

The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis.

OBJECTIVE: Eosinophil predominant inflammation characterises histological features of eosinophilic oesophagitis (EoE). Endoscopy with biopsy is currently the only method to assess oesophageal mucosal inflammation in EoE. We hypothesised that measurements of luminal eosinophil-derived proteins would correlate with oesophageal mucosal inflammation in children with EoE. DESIGN: The Enterotest diagnostic device was used to develop an oesophageal string test (EST) as a minimally invasive clinical device. EST samples and oesophageal mucosal biopsies were obtained from children undergoing upper endoscopy for clinically defined indications. Eosinophil-derived proteins including eosinophil secondary granule proteins (major basic protein-1, eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil peroxidase) and Charcot-Leyden crystal protein/galectin-10 were measured by ELISA in luminal effluents eluted from ESTs and extracts of mucosal biopsies. RESULTS: ESTs were performed in 41 children with active EoE (n=14), EoE in remission (n=8), gastro-oesophageal reflux disease (n=4) and controls with normal oesophagus (n=15). EST measurement of eosinophil-derived protein biomarkers significantly distinguished between children with active EoE, treated EoE in remission, gastro-oesophageal reflux disease and normal oesophagus. Levels of luminal eosinophil-derived proteins in EST samples significantly correlated with peak and mean oesophageal eosinophils/high power field (HPF), eosinophil peroxidase indices and levels of the same eosinophil-derived proteins in extracts of oesophageal biopsies. CONCLUSIONS: The presence of eosinophil-derived proteins in luminal secretions is reflective of mucosal inflammation in children with EoE. The EST is a novel, minimally invasive device for measuring oesophageal eosinophilic inflammation in children with EoE.

Combined activities of secretory phospholipases and eosinophil lysophospholipases induce pulmonary surfactant dysfunction by phospholipid hydrolysis.

BACKGROUND: Surfactant dysfunction is implicated in small airway closure in asthma. Increased activity of secretory phospholipase A(2) (sPLA(2)) in the airways is associated with asthma exacerbations. Phosphatidylcholine, the principal component of pulmonary surfactant that maintains small airway patency, is hydrolyzed by sPLA(2). The lysophosphatidylcholine product is the substrate for eosinophil lysophospholipases. OBJECTIVE: To determine whether surfactant phospholipid hydrolysis by the combined activities of sPLA(2)s and eosinophil lysophospholipases induces surfactant dysfunction. METHODS: The effect of these enzymes on surfactant function was determined by capillary surfactometry. Thin layer chromatography was used to correlate enzyme-induced changes in surfactant phospholipid composition and function. Phosphatidylcholine and its hydrolytic products were measured by using mass spectrometry. RESULTS: Eosinophils express a 25-kd lysophospholipase and group IIA sPLA(2). Phospholipase A(2) alone induced only a small decrease in surfactant function, and 25-kd lysophospholipase alone degraded lysophosphatidylcholine but had no effect on surfactant function. The combined actions of sPLA(2) and lysophospholipase produced dose-dependent and time-dependent losses of surfactant function, concomitant with hydrolysis of phosphatidylcholine and lysophosphatidylcholine. Lysates of AML14.3D10 eosinophils induced surfactant dysfunction, indicating these cells express all the necessary lipolytic activities. In contrast, lysates of blood eosinophils required exogenous phospholipase A(2) to induce maximal surfactant dysfunction. CONCLUSION: The combined activities of sPLA(2)s and eosinophil lysophospholipases are necessary to degrade surfactant phospholipids sufficiently to induce functional losses in surfactant activity as reported in asthma. CLINICAL IMPLICATIONS: The phospholipases and lysophospholipases expressed by eosinophils or other airway cells may represent novel therapeutic targets for blocking surfactant degradation, dysfunction, and peripheral airway closure in asthma.

Charcot-Leyden crystal protein (galectin-10) is not a dual function galectin with lysophospholipase activity but binds a lysophospholipase inhibitor in a novel structural fashion.

Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophil's lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide and p-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys(29) and with Cys(57) near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp(72), in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophil's lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.