Sheena C. Kerr

Roles of epithelial cell-derived periostin in TGF-β activation, collagen production, and collagen gel elasticity in asthma

Periostin is considered to be a matricellular protein with expression typically confined to cells of mesenchymal origin. Here, by using in situ hybridization, we show that periostin is specifically up-regulated in bronchial epithelial cells of asthmatic subjects, and in vitro, we show that periostin protein is basally secreted by airway epithelial cells in response to IL-13 to influence epithelial cell function, epithelial–mesenchymal interactions, and extracellular matrix organization. In primary human bronchial epithelial cells stimulated with periostin and epithelial cells overexpressing periostin, we reveal a function for periostin in stimulating the TGF-β signaling pathway in a mechanism involving matrix metalloproteinases 2 and 9. Furthermore, conditioned medium from the epithelial cells overexpressing periostin caused TGF-β–dependent secretion of type 1 collagen by airway fibroblasts. In addition, mixing recombinant periostin with type 1 collagen in solution caused a dramatic increase in the elastic modulus of the collagen gel, indicating that periostin alters collagen fibrillogenesis or cross-linking and leads to stiffening of the matrix. Epithelial cell-derived periostin in asthma has roles in TGF-β activation and collagen gel elasticity in asthma.

CXCR4 expression reflects tumor progression and regulates motility of bladder cancer cells

Transitional cell carcinoma of the urinary bladder remains life threatening due to the high occurrence of metastases. Emerging evidence suggests that chemokines and their receptors play a critical role in tumor metastases. In our study, we performed a systematic analysis of the mRNA and protein expression levels of all 18 chemokine receptors in normal urothelium and bladder cancer. CXCR4 was the only chemokine receptor whose mRNA expression level was upregulated in bladder cancer cell lines as well as in invasive and locally advanced bladder cancer tissue samples (pT2–pT4). In contrast, superficial bladder tumors (pTa and pT1) displayed low CXCR4 expression levels and normal urothelial cells were negative for CXCR4. Immunohistochemistry of a bladder cancer tissue microarray (TMA) confirmed that a subgroup of invasive bladder cancers revealed a high CXCR4 protein expression, while superficial bladder tumors showed low immunoreactivity. To investigate the functional significance of CXCR4 expression, we performed migration and invasion assays. Exposure of CXCR4‐positive bladder cancer cells to CXCL12 in a Boyden chamber type assay provoked a significant increase in migration as well as invasion across a Matrigel barrier. Enhanced migration and invasion were inhibited by a CXCR4‐specific blocking antibody. In contrast, normal urothelial cells did not respond to CXCL12 and lacked chemotactic migration. In conclusion, bladder cancer cells express CXCR4 progressively with advanced tumorigenesis and this receptor interacts with CXCL12 to mediate tumor chemotaxis and invasion through connective tissue. These properties identify CXCR4 as a potential target for the attenuation of bladder cancer metastases.

A soluble fucose-specific lectin from Aspergillus fumigatus conidia - Structure, specificity and possible role in fungal pathogenicity

Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many other pathogens, it is able to produce lectins that may be involved in the host-pathogen interaction. We focused on the lectin AFL, which was prepared in recombinant form and characterized. Its binding properties were studied using hemagglutination and glycan array analysis. We determined the specificity of the lectin towards l-fucose and fucosylated oligosaccharides, including α1-6 linked core-fucose, which is an important marker for cancerogenesis. Other biologically relevant saccharides such as sialic acid, d-mannose or d-galactose were not bound. Blood group epitopes of the ABH and Lewis systems were recognized, LeY being the preferred ligand among others. To provide a correlation between the observed functional characteristics and structural basis, AFL was crystallized in a complex with methyl-α,l-selenofucoside and its structure was solved using the SAD method. Six binding sites, each with different compositions, were identified per monomer and significant differences from the homologous AAL lectin were found. Structure-derived peptides were utilized to prepare anti-AFL polyclonal antibodies, which suggested the presence of AFL on the Aspergillus’ conidia, confirming its expression in vivo. Stimulation of human bronchial cells by AFL led to IL-8 production in a dose-dependent manner. AFL thus probably contributes to the inflammatory response observed upon the exposure of a patient to A. fumigatus. The combination of affinity to human epithelial epitopes, production by conidia and pro-inflammatory activity is remarkable and shows that AFL might be an important virulence factor involved in an early stage of A. fumigatus infection.

Hexaphenylbenzene as a Rigid Template for the Straightforward Syntheses of “Star-Shaped” Glycodendrimers

Original glycodendrimers emanating from propargylated hexaphenylbenzene cores and containing up to 54 peripheral sugar ligands have been synthesized by Cu(I)-catalyzed [1,3]-dipolar cycloadditions using both convergent and divergent approaches.

FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

Intelectin-1 Is a Prominent Protein Constituent of Pathologic Mucus Associated with Eosinophilic Airway Inflammation in Asthma

To the Editor: Intelectin-1 (ITLN-1) is an epithelial cell protein that is up-regulated in asthma (1). ITLN-1 is a pleotropic adipokine (also known as omentin-1) with roles in the gut ranging from host defense against pathogenic bacteria to promotion of insulin-stimulated glucose uptake (2–4). The host defense roles of ITLN-1 may result from its ability to bind structures expressed by microorganisms in a carbohydrate-dependent manner (5). ITLN-1 is also a binding partner for lactoferrin (6), and ITLN-1 may cooperate with lactoferrin in host defense (6, 7). Little is known about the function of ITLN-1 in human asthma. One possibility is that it participates in pathways of inflammation downstream of IL-13 (1). Indeed, studies in a mouse model of asthma suggest that ITLN-1 mediates IL-13–induced monocyte chemotactic protein-1 and -3 production in epithelial cells (8). Another possibility is that ITLN-1 is a component of airway mucus and contributes to pathologic mucus gel formation in disease. Supporting this possibility are studies in the gastrointestinal tract showing that ITLN-1 is a goblet cell protein that is secreted with mucus into the intestinal lumen (9). In addition, other studies in the intestine have suggested mucin–intelectin interactions that could alter the biophysical properties of mucus (10). Some of the results of these studies have been previously reported in the form of an abstract (11) Because mucus pathology causes mucus plugging and airway occlusion (12, 13), especially in fatal asthma (14), we set out to determine if ITLN-1 is a component of pathologic mucus in acute asthma. We first immunostained lung tissue sections from cases of fatal asthma and found prominent ITLN-1 immunostaining in the pathologic mucus plugs that occlude the airways (Figures 1A–1C). The cellular source of the ITLN-1 appears to be goblet cells (Figure 1C). We next measured ITLN-1 protein in sputum from 11 patients with acute severe asthma and two control groups (35 subjects with chronic stable asthma and 11 healthy control subjects) (Table 1). We found that ITLN-1 protein levels in the subgroup of patients with asthma in exacerbation were significantly higher than in stable asthma and in healthy control subjects (Figure 1D). We also noted that the increase in ITLN-1 in acute asthma was driven by the subgroup with increased sputum eosinophils (>2%) (Figure 1E), a finding that is consistent with ITLN-1’s regulation by IL-13 in airway epithelial cells (1). ITLN-1 up-regulation is thus a feature of “Th2-high” asthma, and the known pathologic characteristics of this disease endotype can be extended to include high ITLN-1 protein concentrations in mucus forming during disease exacerbations. Figure 1. Intelectin-1 (ITLN-1) protein in airway biospecimens and binding of ITLN-1 to airway mucins and lactoferrin. Sections of lung tissue from lungs of patients with fatal asthma were stained with an anti–ITLN-1 antibody or peptide blocking control. ... Table 1: Subject Characteristics The prominent immunostaining for ITLN-1 in mucus plugs in fatal asthma and the high concentrations of ITLN-1 in sputum in acute severe asthma prompted us to explore if ITLN-1 can bind to human airway mucins. ITLN-1 is a lectin with known specificity for galactosyl structures, especially the galactofuranosyl sugars expressed by microorganisms (5). To determine if ITLN-1 binds to human airway mucin glycans, we developed a plate-based binding assay using high-molecular-weight mucin preparations that we purified from induced sputum samples from subjects with chronic stable asthma (“mucin study”; Table 1). Specifically, we used biotinylated recombinant ITLN-1 to probe mucin coated on microtiter plates (see online supplement). Biotinylated jacalin, a tetrameric plant seed lectin with specificity for galactose, was used as a positive control. Although jacalin showed binding to mucin, ITLN-1 did not (Figure 1F). It is possible that ITLN-1 cannot recognize the pyranosyl forms of galactose in human mucin, but another possibility is that mucin glycans prevent binding through steric hindrance. It could also be that the plate assay is suboptimal for measuring ITLN-1 binding to mucin because other proteins or cofactors involved in an ITLN-1–mucin interaction in vivo are not represented in vitro. Because ITLN-1 has been characterized as the lactoferrin receptor (6, 7), we considered if ITLN-1 interacts with lactoferrin in airway mucus in acute asthma. We found that lactoferrin levels in sputum from patients with acute severe asthma are significantly higher than in control samples (Figure 1G). Notably, the concentration of lactoferrin ranged from 500 to 1,000 μg/ml in some of these sputum samples, a 1,000-fold higher concentration than ITLN-1. This large amount of lactoferrin in asthmatic mucus may bind and concentrate ITLN-1 in mucus. To examine the binding of ITLN-1 to lactoferrin, we used a plate-binding assay similar to the one we used for mucin-ITLN-1 binding. In this way, we found that biotinylated ITLN-1 binds avidly to immobilized lactoferrin (Figure 1H). This binding was inhibited by heparin, suggesting a charge-based interaction between ITLN-1 and lactoferrin’s basic N-terminal region (15), and increased by methyl galactofuranoside. Galactofuranoside is found in many microbial polysaccharides and is recognized as a preferred glycan ligand for ITLN-1 (7, 16). Our data suggest that ITLN-1 binding to galactofuranosyl residues on microorganisms might improve its ability to bind lactoferrin and target it to regions of high microorganism burden. We conclude that ITLN-1 is a prominent protein component of pathologic mucus in fatal asthma and in acute severe asthma, especially in the context of eosinophilic airway inflammation. The binding of ITLN-1 to lactoferrin is increased by galactofuranoside providing a mechanism by which ITLN-1 can cooperate with lactoferrin to defend against microbes.

Endoglycan, a Member of the CD34 Family of Sialomucins, Is a Ligand for the Vascular Selectins

The interactions of the selectin family of adhesion molecules with their ligands are essential for the initial rolling stage of leukocyte trafficking. Under inflammatory conditions, the vascular selectins, E- and P-selectin, are expressed on activated vessels and interact with carbohydrate-based ligands on the leukocyte surface. While several ligands have been characterized on human T cells, monocytes and neutrophils, there is limited information concerning ligands on B cells. Endoglycan (EG) together with CD34 and podocalyxin comprise the CD34 family of sialomucins. We found that EG, previously implicated as an L-selectin ligand on endothelial cells, was present on human B cells, T cells and peripheral blood monocytes. Upon activation of B cells, EG increased with a concurrent decrease in PSGL-1. Expression of EG on T cells remained constant under the same conditions. We further found that native EG from several sources (a B cell line, a monocyte line and human tonsils) was reactive with HECA-452, a mAb that recognizes sialyl Lewis X and related structures. Moreover, immunopurified EG from these sources was able to bind to P-selectin and where tested E-selectin. This interaction was divalent cation-dependent and required sialylation of EG. Finally, an EG construct supported slow rolling of E- and P-selectin bearing cells in a sialic acid and fucose dependent manner, and the introduction of intact EG into a B cell line facilitated rolling interactions on a P-selectin substratum. These in vitro findings indicate that EG can function as a ligand for the vascular selectins.