N-Glycans differentially regulate eosinophil and neutrophil recruitment during allergic airway inflammation

Abstract

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.

FIGURE 1.

Allergen-induced airway inflammation is reduced in Mgat5^−/− mice. A, BALF total and differential cell counts in control and allergen-challenged WT and Mgat5^−/− mice (10–14 mice per group) by microscopic evaluation of Hema 3-stained cytocentrifuged slides. Combined data from experiments repeated at least three times is shown. Eos, eosinophils; Monos/Macros, monocytes/macrophages; Lymphs, lymphocytes; Neu, neutrophils. B, cellular infiltration of lung sections after H&E staining. Representative images (magnification, ×100) are shown (n = 5 mice per group). C, infiltration of lung tissue by eosinophils after staining with rat mAb against murine MBP. Representative images (magnification, ×200) are shown for allergen-challenged groups (left). Very few to no MBP-positive cells were detected in controls (n = 4 mice per group). Representative histogram of PHA-L binding by gated BALF eosinophils from allergen-challenged WT and Mgat5^−/− mice by flow cytometry (right). WT and Mgat5^−/− BALF eosinophils exhibited similar binding to BSA. Only WT BALF eosinophil binding to BSA is shown (n = 6 mice per group). D, mucus production in airways after PAS staining. PAS-positive cells in airways of relatively similar size were quantitated (4–20 airways/slide) by light microscopy and expressed as a percentage of the total number of epithelial cells per airway. FIZZ1 (E) and intelectin-1 (F) expression in lung sections by immunohistology. FIZZ1- and intelectin-1-positive area was quantitated by ImageJ analysis of captured images of 5–10 fields per slide (n = 4–5 mice per group in D–F). Control mice in each group showed little or no PAS and intelectin-1 staining. Representative microscopic images (magnification, ×200) of allergen-challenged groups are shown below bars in D–F. Data represent mean ± S.E. in A, D, E, and F. *, p < 0.05 for allergen-challenged Mgat5^−/− versus WT mice.

FIGURE 2.

MGAT5-modified N-glycans are essential for eosinophil trafficking and survival. Rolling (A) and adhesion (B) of WT and Mgat5^−/− eosinophils on immobilized VCAM-1 or galectin-3 (Gal-3) under conditions of flow. Rolling on PBS was considered as background. C, chemotaxis of WT and Mgat5^−/− eosinophils in response to eotaxin (100 nm) or medium alone (control) in vitro. Combined data of two independent experiments performed in triplicate for each condition with eosinophils from two different mice for each group is shown for A–C. D, basal [Ca²⁺]_i levels in WT and Mgat5^−/− eosinophils by digital videofluorescence imaging using the Ca2⁺ indicator dye Fura-2 AM. Combined data of three pooled experiments with Mgat5^−/− and WT eosinophils (n = 270–340 cells) from three different mice. E, eosinophil survival evaluated by flow cytometry based on FITC-annexin V binding by gated eosinophils. Combined data of three experiments with eosinophils from three different mice for each group is shown. Data represent mean ± S.E. in A–E. *, p < 0.05 for comparison of WT versus Mgat5^−/− eosinophils.

FIGURE 3.

Allergen-challenged Mgat5^−/− mice exhibit increased AHR and develop lung neutrophilia. A, AHR in control and allergen-challenged WT and Mgat5^−/− mice assessed by whole body plethysmography. The enhanced pause in breathing after MCh was expressed as a percentage of baseline enhanced pause with saline (n = 8 mice per group). B, Gr-1 expression in lung tissue lysates of control and allergen-challenged mice by densitometry of Western blots normalized against β-actin expression (n = 4–5 per group). C, representative images of lung tissue neutrophils in allergen-challenged WT and Mgat5^−/− mice by confocal microscopy after staining with mAb NIMP-R14 (magnification, ×600). No NIMP-R14-positive cells were detected in control lungs from both groups (n = 4–5 per group). D, KC levels in lung tissue lysates of control and allergen-challenged WT and Mgat5^−/− mice by flow cytometry (n = 4–6 per group). Data represent mean ± S.E. in A, C, and E. *, p < 0.05 versus respective control; and #, p < 0.05 between allergen-challenged groups for A; **, p < 0.05 versus WT OVA in B and D.

FIGURE 4.

MGAT5 deficiency enhances neutrophilic inflammation in response to LPS and TG. BALF cell counts (A) and peripheral blood neutrophils (B) in LPS-exposed WT and Mgat5^−/− mice after Hema 3 staining. C, differential cell counts in the peritoneal fluid of TG-exposed WT and Mgat5^−/− mice. Neu, neutrophils; Monos, monocytes; Lymphs, lymphocytes. D, number of labeled cells in the peritoneal fluid of TG-exposed WT mice 6 h after transfer for neutrophils and 2 h after transfer for eosinophils (Eos). Combined data of three experiments is shown in each case (n = 4 mice per group in A and B and n = 3 mice per group in C and D). Data represent mean ± S.E. *, p < 0.05 for Mgat5^−/− versus WT in A, C, and D. **, p < 0.05 for post-LPS versus pre-LPS in Mgat5^−/− mice in B.

FIGURE 5.

Mgat5^−/− neutrophils exhibit increased adhesion and migration along with elevated CXCR2 expression. Rolling (A) and adhesion (B) of WT and Mgat5^−/− neutrophils on immobilized P-selectin under conditions of flow. Adhesion is expressed as fold increase over background (adhesion to PBS). Combined data of three independent experiments performed in duplicate for each condition are shown for A and B. C, quantitation of WT and Mgat5^−/− neutrophils adhered to P-selectin under static conditions. Adherent cells in six fields were counted for each cell type, and combined data of two independent experiments are shown. D, migration of WT and Mgat5^−/− neutrophils in response to KC (100 nm) or medium alone (control). Combined data of two experiments performed in triplicate for each condition with neutrophils from two different mice for each group is shown. E, surface expression of CXCR2 (CD182) on WT and Mgat5^−/− neutrophils by flow cytometry with PerCP/Cy5.5 anti-mouse CD182 (5 μg/ml) before and after activation with KC (100 ng/ml). Histograms shown are representative of three independent experiments. F, relative expression of CXCR2 on WT versus Mgat5^−/− neutrophils by flow cytometry after activation with KC as percent cells positive for CXCR2. Combined data of three independent experiments with neutrophils from three different mice for each group is shown. G, basal and KC-induced [Ca²⁺]_i levels in WT and Mgat5^−/− neutrophils by digital videofluorescence imaging with Fura-2 AM. Combined data of three pooled experiments with Mgat5^−/− and WT neutrophils (∼500 cells for each group) from three different mice are shown. H, neutrophil survival evaluated flow cytometry based on FITC-annexin V binding by gated neutrophils. Combined data of three independent experiments with neutrophils from three different mice are shown. Data represent mean ± S.E. in A–D and F–H. *, p < 0.05 for Mgat5^−/− versus WT neutrophils in B, C, D, and F, and versus respective control in A (rolling on PBS) and G (without KC). #, p < 0.05 for Mgat5^−/− versus WT neutrophils with KC treatment in G.