Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Abstract

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661-1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of alpha2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV(-/-) neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV(-/-) neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV(-/-) leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV(-/-) leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of alpha2,3-linked sialic acid-specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.

Figure 1.

Leukocyte adhesion during inflammation in vivo. (A) Number of adherent leukocytes (mean ± SEM per mm² of surface area) in unstimulated mouse cremaster muscle venules of WT control and ST3Gal-IV^−/− mice. (B) Increase in the number of adherent leukocytes (mean ± SEM per mm² of surface area) 3 min after systemic injection of 600 ng CXCL1 or CXCL8 in WT control, ST3Gal-IV^−/−, WT control (plus 4 μg PTx), ST3Gal-IV^−/− (plus 4 μg PTx), and CXCR2^bm−/− mice. PTx was injected 3 h before cremaster muscle exteriorization. (C) Leukocyte adhesion (mean ± SEM) was observed in 3-h TNF-α–treated cremaster muscle venules of WT control, ST3Gal-IV^−/−, and CXCR2^bm−/− mice. Additionally, pretreatment of TNF-α–treated WT control, ST3Gal-IV^−/−, and CXCR2^bm−/− mice was conducted with 4 μg PTx and/or 100 μg E-selectin mAb 9A9 5 min before TNF-α injection. Data in A–C were obtained from at least three independent experiments per group. *, P < 0.05 versus WT control mice (A and C); *, P < 0.05 versus WT control mice treated with CXCL1 or CXCL8, respectively (B). Videos 1 and 2 are available at http://www.jem.org/cgi/content/full/jem.20070846/DC1.

Figure 2.

Cremaster muscle whole mount. Giemsa-stained whole-mounts of TNF-α–treated cremaster muscles of WT, ST3Gal-IV^−/−, and CXCR2^bm−/− mice were analyzed for the number of intravascular (A) and perivascular (B) leukocytes (mean ± SEM per mm² surface area). In addition, two typical micrographs are presented illustrating intra- and perivascular leukocyte distribution in TNF-α–treated cremaster muscle whole mounts of WT control mice (C) and ST3Gal-IV^−/− mice pretreated with E-selectin blocking mAb 9A9 (D). Data in A and B were obtained from at least three independent experiments per group. *, P < 0.05 versus WT mice. Bars, 50 μm.

Figure 3.

Thioglycollate-induced peritonitis. Peritoneal neutrophil influx was assessed 4 h after injection of 4% thioglycollate into WT (n = 7), ST3Gal-IV^−/− (n = 9), WT (plus 4 μg PTx; n = 6), ST3Gal-IV^−/− (plus 4 μg PTx; n = 5), and CXCR2^bm−/− (n = 3) mice. The number of neutrophils (×10⁶; mean ± SEM) was assessed from Turks-stained peritoneal lavage samples. *, P < 0.05 versus WT control mice.

Figure 4.

CXCR2 expression and ligand binding of ST3Gal-IV^−/− leukocytes. (A) Surface expression of CXCR2 on GR-1–positive leukocytes of ST3Gal-IV^−/− and WT mice was analyzed by flow cytometry. Leukocytes were incubated with an allophycocyanin-labeled GR-1 mAb, a PE-labeled anti-CXCR2 mAb, and a PE-labeled isotype control. (B) For ligand binding studies, leukocytes from the peripheral blood of WT (n = 4), ST3Gal-IV^−/− (n = 3), and CXCR2^bm−/− (n = 3) mice were incubated with different dilutions of CF-CXCL8 and subsequently analyzed for bound fluorescence by flow cytometry. Peripheral blood leukocytes from WT mice were treated with 100 mU/ml neuraminidase for 60 min and investigated for CF-CXCL8 binding. Data are given as mean ± SD. P < 0.05 for WT versus ST3Gal-IV^−/−, CXCR2^bm−/−, and WT + Neu for 30 and 90 nM CF-CXCL8, respectively. MFI, mean fluorescence intensity.

Figure 5.

Adhesion in the flow chamber and transmigration of ST3Gal-IV^−/− neutrophils in vitro. The number of rolling and adherent leukocytes from ST3Gal-IV^−/− and WT mice was determined in blood-perfused microflow chambers. (A) Rolling and (B) adherent leukocytes in autoperfused flow chambers (5.94 dynes/cm²) coated with 20 μg/ml P-selectin and 15 μg/ml ICAM-1 with or without 15 μg/ml CXCL1. Data in A and B are presented as mean ± SEM from at least four mice and four flow chambers. *, P < 0.05 versus WT mice (B). (C) The percentage of transmigrated neutrophils from WT control mice, ST3Gal-IV^−/− mice, and WT control mice pretreated with neuraminidase was assessed in a transwell assay in which transwells were coated with an immortalized endothelial cell line, b.End5. In addition, transmigration of WT control neutrophils through a neuraminidase-pretreated b.End5 monolayer was analyzed. Data in C were determined from at least three independent experiments per group. *, P < 0.05 versus untreated WT control neutrophils (C). FOV, field of view.

Figure 6.

Effect of enzymatic desialylation on chemokine binding and function of human CXCR2. (A) Cell lysates of CXCR2-transfected and untransfected HEK293 cells were incubated with an mAb to CXCR2, followed by precipitation with protein A. Precipitates were treated with 20 U/ml neuraminidase for 60 min at 37°C, or were left untreated and analyzed by Western blotting for binding of biotinylated anti-CXCR2 antibody or biotinylated MAL-II, respectively. (B) Isolated human neutrophils were treated with the indicated dosages of neuraminidase for 30 min at 37°C, washed, and subsequently assayed for binding of ¹²⁵I-labeled CXCL7 and ¹²⁵I-labeled CXCL4. Specifically bound radioactivity was expressed as the percentage of WT control cells receiving no enzyme treatment. (C) Scatchard plot for CXCL7 binding to untreated and neuraminidase-treated (20 U/ml) human neutrophils. The binding data were curve fit to determine affinity constants for high and low affinity binding sites. (D) Neutrophils were treated with the indicated dosages of neuraminidase for 30 min at 37°C, washed, and subsequently assayed for elastase release in response to increasing concentrations of CXCL7, CXCL8, and fMLP. The residual responsiveness of neuraminidase-treated neutrophils was expressed as the percentage of that determined for untreated cells. Data in A–D represent means ± SD determined in three different experiments, each with blood from a different donor.