Nooshin S. Bahaie

Galectin-3 Functions as an Adhesion Molecule to Support Eosinophil Rolling and Adhesion under Conditions of Flow

Allergic inflammation involves the mobilization and trafficking of eosinophils to sites of inflammation. Galectin-3 (Gal-3) has been shown to play a critical role in eosinophil recruitment and airway allergic inflammation in vivo. The role played by Gal-3 in human eosinophil trafficking was investigated. Eosinophils from allergic donors expressed elevated levels of Gal-3 and demonstrated significantly increased rolling and firm adhesion on immobilized VCAM-1 and, more surprisingly, on Gal-3 under conditions of flow. Inhibition studies with specific mAbs as well as lactose demonstrated that: 1) eosinophil-expressed Gal-3 mediates rolling and adhesion on VCAM-1; 2) α4 integrin mediates eosinophil rolling on immobilized Gal-3; and 3) eosinophil-expressed Gal-3 interacts with immobilized Gal-3 through the carbohydrate recognition domain of Gal-3 during eosinophil trafficking. These findings were further confirmed using inflamed endothelial cells. Interestingly, Gal-3 was found to bind to α4 integrin by ELISA, and the two molecules exhibited colocalized expression on the cell surface of eosinophils from allergic donors. These findings suggest that Gal-3 functions as a cell surface adhesion molecule to support eosinophil rolling and adhesion under conditions of flow.

Allergen-Induced Airway Remodeling Is Impaired in Galectin-3–Deficient Mice

The role played by the β-galactoside–binding lectin galectin-3 (Gal-3) in airway remodeling, a characteristic feature of asthma that leads to airway dysfunction and poor clinical outcome in humans, was investigated in a murine model of chronic allergic airway inflammation. Wild-type (WT) and Gal-3 knockout (KO) mice were subjected to repetitive allergen challenge with OVA up to 12 wk, and bronchoalveolar lavage fluid (BALF) and lung tissue collected after the last challenge were evaluated for cellular features associated with airway remodeling. Compared to WT mice, chronic OVA challenge in Gal-3 KO mice resulted in diminished remodeling of the airways with significantly reduced mucus secretion, subepithelial fibrosis, smooth muscle thickness, and peribronchial angiogenesis. The higher degree of airway remodeling in WT mice was associated with higher Gal-3 expression in the BALF as well as lung tissue. Cell counts in BALF and lung immunohistology demonstrated that eosinophil infiltration in OVA-challenged Gal-3 KO mice was significantly reduced compared with that WT mice. Evaluation of cellular mediators associated with eosinophil recruitment and airway remodeling revealed that levels of eotaxin-1, IL-5, IL-13, found in inflammatory zone 1, and TGF-β were substantially lower in Gal-3 KO mice. Finally, leukocytes from Gal-3 KO mice demonstrated decreased trafficking (rolling) on vascular endothelial adhesion molecules compared with that of WT cells. Overall, these studies demonstrate that Gal-3 is an important lectin that promotes airway remodeling via airway recruitment of inflammatory cells, specifically eosinophils, and the development of a Th2 phenotype as well as increased expression of eosinophil-specific chemokines and profibrogenic and angiogenic mediators.

ORMDL3 promotes eosinophil trafficking and activation via regulation of integrins and CD48

ORM (yeast)-Like protein isoform 3 (ORMDL3) has recently been identified as a candidate gene for susceptibility to asthma; however the mechanisms by which it contributes to asthma pathogenesis are not well understood. Here we demonstrate a functional role for ORMDL3 in eosinophils in the context of allergic inflammation. Eosinophils recruited to the airways of allergen-challenged mice express ORMDL3. ORMDL3 expression in bone marrow eosinophils is localized in the endoplasmic reticulum and is induced by IL-3 and eotaxin-1. Over-expression of ORMDL3 in eosinophils causes increased rolling, distinct cytoskeletal rearrangement, ERK (1/2) phosphorylation and nuclear translocation of NF-κB. Knock-down of ORMDL3 significantly inhibits activation-induced cell shape changes, adhesion and recruitment to sites of inflammation in vivo, combined with reduced expression of CD49d and CD18. Additionally, ORMDL3 regulates IL-3-induced expression of CD48 and CD48-mediated eosinophil degranulation. These studies show that ORMDL3 regulates eosinophil trafficking, recruitment and degranulation, further elucidating a role for this molecule in allergic asthma and potentially other eosinophilic disorders.

Deficiency of Endothelial Heparan Sulfates Attenuates Allergic Airway Inflammation

The effect of targeted inactivation of the gene encoding N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in the biosynthesis of heparan sulfate (HS) chains, on the inflammatory response associated with allergic inflammation in a murine model of OVA-induced acute airway inflammation was investigated. OVA-exposed Ndst1f/fTekCre+ (mutant) mice deficient in endothelial and leukocyte Ndst1 demonstrated significantly decreased allergen-induced airway hyperresponsiveness and inflammation characterized by a significant reduction in airway recruitment of inflammatory cells (eosinophils, macrophages, neutrophils, and lymphocytes), diminished IL-5, IL-2, TGF-β1, and eotaxin levels, as well as decreased expression of TGF-β1 and the angiogenic protein FIZZ1 (found in inflammatory zone 1) in lung tissue compared with OVA-exposed Ndst1f/fTekCre− wild-type littermates. Furthermore, murine eosinophils demonstrated significantly decreased rolling on lung endothelial cells (ECs) from mutant mice compared with wild-type ECs under conditions of flow in vitro. Treatment of wild-type ECs, but not eosinophils, with anti-HS Abs significantly inhibited eosinophil rolling, mimicking that observed with Ndst1-deficient ECs. In vivo, trafficking of circulating leukocytes in lung microvessels of allergen-challenged Ndst1-deficient mice was significantly lower than that observed in corresponding WT littermates. Endothelial-expressed HS plays an important role in allergic airway inflammation through the regulation of recruitment of inflammatory cells to the airways by mediating interaction of leukocytes with the vascular endothelium. Furthermore, HS may also participate by sequestering and modulating the activity of allergic asthma-relevant mediators such as IL-5, IL-2, and TGF-β1.

Regulation of Eosinophil Trafficking by SWAP-70 and Its Role in Allergic Airway Inflammation

Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70–deficient (Swap-70−/−) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70−/− eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70−/− eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70−/− eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70−/− mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.

ADAM17 cleaves CD16b (FcγRIIIb) in human neutrophils

CD16b (FcγRIIIb) is exclusively expressed by human neutrophils and binds IgG in immune complexes. Cell surface CD16b undergoes efficient ectodomain shedding upon neutrophil activation and apoptosis. Indeed, soluble CD16b is present at high levels in the plasma of healthy individuals, which appears to be maintained by the daily turnover of apoptotic neutrophils. At this time, the principal protease responsible for CD16b shedding is not known. We show that CD16b plasma levels were significantly decreased in patients administered a selective inhibitor targeting the metalloproteases ADAM10 and ADAM17. Additional analysis with inhibitors selective for ADAM10 or ADAM17 revealed that only inhibition of ADAM17 significantly blocked the cleavage of CD16b following neutrophil activation and apoptosis. CD16b shedding by ADAM17 was further demonstrated using a unique ADAM17 function-blocking mAb and a cell-based ADAM17 reconstitution assay. Unlike human CD16, however, mouse CD16 did not undergo efficient ectodomain shedding upon neutrophil stimulation or apoptosis, indicating that this mechanism cannot be modeled in normal mice. Taken together, our findings are the first to directly demonstrate that ADAM17 cleaves CD16 in human leukocytes.

The p110δ subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice

Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.

N-Glycans Differentially Regulate Eosinophil and Neutrophil Recruitment during Allergic Airway Inflammation

Background: Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment. Results: Deficiency of MGAT5 causes attenuation of allergen-induced eosinophilia and Th2 cytokines but increases neutrophilic inflammation and airway hyperreactivity. Conclusion: Recruitment of eosinophils and neutrophils is differentially regulated by MGAT5-modified N-glycans during airway inflammation. Significance: This study demonstrates a significant role for N-glycans in the development of allergic airway inflammation and asthma. Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-d-mannoside β1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5−/−) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5−/− eosinophils. Interestingly, allergen-challenged Mgat5−/− mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5−/− mice. Furthermore, there was significantly increased recruitment of infused Mgat5−/− neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5−/− neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.

Regulation of Serotonin-Induced Trafficking and Migration of Eosinophils

Association of the neurotransmitter serotonin (5-HT) with the pathogenesis of allergic asthma is well recognized and its role as a chemoattractant for eosinophils (Eos) in vitro and in vivo has been previously demonstrated. Here we have examined the regulation of 5-HT-induced human and murine Eos trafficking and migration at a cellular and molecular level. Eos from allergic donors and bone marrow-derived murine Eos (BM-Eos) were found to predominantly express the 5-HT2A receptor. Exposure to 5-HT or 2,5-dimethoxy-4-iodoamphetamine (DOI), a 5-HT2A/C selective agonist, induced rolling of human Eos and AML14.3D10 human Eos-like cells on vascular cell adhesion molecule (VCAM)-1 under conditions of flow in vitro coupled with distinct cytoskeletal and cell shape changes as well as phosphorylation of MAPK. Blockade of 5-HT2A or of ROCK MAPK, PI3K, PKC and calmodulin, but not Gαi-proteins, with specific inhibitors inhibited DOI-induced rolling, actin polymerization and changes in morphology of VCAM-1-adherent AML14.3D10 cells. More extensive studies with murine BM-Eos demonstrated the role of 5-HT in promoting rolling in vivo within inflamed post-capillary venules of the mouse cremaster microcirculation and confirmed that down-stream signaling of 5-HT2A activation involves ROCK, MAPK, PI3K, PKC and calmodulin similar to AML14.3D10 cells. DOI-induced migration of BM-Eos is also dependent on these signaling molecules and requires Ca2+. Further, activation of 5-HT2A with DOI led to an increase in intracellular Ca2+ levels in murine BM-Eos. Overall, these data demonstrate that 5-HT (or DOI)/5-HT2A interaction regulates Eos trafficking and migration by promoting actin polymerization associated with changes in cell shape/morphology that favor cellular trafficking and recruitment via activation of specific intracellular signaling molecules (ROCK, MAPK, PI3K and the PKC-calmodulin pathway).

Regulation of CXCR2 expression and function by a disintegrin and metalloprotease-17 (ADAM17)

The chemokine receptor CXCR2 is expressed at high levels on circulating neutrophils and is critical for directing their migration to sites of inflammation. CXCR2 surface levels are rapidly modulated by 2 mechanisms—cell internalization and recycling upon ligand binding—and by a metalloprotease activity following overt neutrophil activation by nonligand stimuli. The latter process has only been described in human neutrophils, and essentially, nothing is known about its functional relevance and the specific protease involved. We show that targeting ADAM17 in mouse and human neutrophils blocks CXCR2 down‐regulation induced by nonligand stimuli but not by chemokine ligands. This was determined by use of a selective ADAM17 inhibitor, an ADAM17 function‐blocking antibody, and ADAM17 gene‐targeted mice. CXCR2 is known to undergo a marked down‐regulation during various inflammatory disorders, and this is associated with impaired neutrophil recruitment. We show that blocking ADAM17 activity reduced CXCR2 down‐regulation on circulating neutrophils and enhanced their recruitment during acute inflammation, which was reversed by a CXCR2 inhibitor. Taken together, our findings demonstrate that unlike CXCR2 internalization, ADAM17 induction down‐regulates the receptor in an irreversible manner and may serve as a master switch in controlling CXCR2 function, but may also contribute to neutrophil dysfunction during excessive inflammation.