Deficiency of the sialyltransferase St3Gal4 reduces Ccl5-mediated myeloid cell recruitment and arrest: short communication

Abstract

Rationale: Sialylation by α2,3-sialyltransferases has been shown to be a crucial glycosylation step in the generation of functional selectin ligands. Recent evidence suggests that sialylation also affects the binding of chemokines to their corresponding receptor.

Objective: Because the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of neutrophils and monocytes, we here investigated the role of α2,3-sialyltransferase IV (ST3Gal-IV) in Ccl5- and Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis.

Methods and results: St3Gal4-deficient myeloid cells showed a reduced binding of Ccl5 and an impaired Ccl5-triggered integrin activation. Correspondingly, Ccl5-induced arrest on tumor necrosis factor-α-stimulated endothelium was almost completely abrogated, as observed in flow chamber adhesion assays and during ex vivo perfusion or intravital microscopy of carotid arteries. Moreover, Ccl5-triggered neutrophil and monocyte extravasation into the peritoneal cavity was severely reduced in St3Gal4(-/-) mice. In contrast, St3Gal4 deficiency did not significantly affect Ccl2 binding and only marginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accumulation in atherogenesis, and the importance of Ccl5 chemokine receptors mediating myeloid cell recruitment to atherosclerotic vessels, St3Gal4 deficiency drastically reduced the size, stage, and inflammatory cell content of atherosclerotic lesions in Apoe(-/-) mice on high-fat diet.

Conclusions: In summary, these findings identify ST3Gal-IV as a promising target to reduce inflammatory leukocyte recruitment and arrest.

Keywords: atherosclerosis; beta-galactoside alpha-2,3-sialyltransferase.

Figure 1. St3Gal4-deficient leukocytes display reduced Ccl5-induced integrin activation and flow arrest

A and B, Ccl5- and Ccl2-induced (2 μg/mL; 5 min) integrin activation in monocytes (A) and neutrophils (B) from St3Gal4^+/+ and St3Gal4^−/− mice, as quantified by the binding of Icam1 and Vcam1. n=5 to 6. MFI indicates mean fluorescence intensity. C, Adhesion of perfused monocytes (left) and neutrophils (right) on tumor necrosis factor-α–activated (10 ng/mL; 4 h), SV40-transformed mouse endothelial cells (SVECs) after pretreatment of leukocytes or SVECs with Ccl5 (2 μg/mL; 10 min). n=6 to 12. D, Intraperitoneal recruitment of monocytes (left) and neutrophils (right) 4 h after intraperitoneal injection of 5 μg Ccl5. n=11 to 16. A to D, Graphs represent mean±SD; Mann–Whitney test (A and B) or 1-way ANOVA with Tukey multiple comparison test (C and D). *P<0.05; **P<0.01; ***P<0.001.

Figure 2. Reduced Ccl5-induced adhesion of St3Gal4^−/− leukocytes on mouse carotid arteries

A and B, Ex vivo flow adhesion of Ccl5-pretreated (2.5 μg/mL; 10 min) leukocytes on tumor necrosis factor-α (Tnfα)-stimulated (20 ng/mL; 4 h) carotids from St3Gal4^+/+ (A) and St3Gal4^−/− (B) mice. n=5 to 7; Mann–Whitney test. C and D, Intravital microscopy of leukocyte adhesion to Tnfα–stimulated carotid arteries of St3Gal4^+/+ and St3Gal4^−/− mice, after leukocyte labeling for CD11b (top, labels all myeloid cells), Ly6G (middle, labels neutrophils), or Ly6C (bottom, labels monocytes). n=6 to 14; t test. A to D, Graphs represent mean±SD. *P<0.05; ***P<0.001.

Figure 3. St3Gal4 deficiency reduces atherosclerosis

Quantification of atherosclerotic lesions in the aortic arch (A) and aortic root (B) of St3Gal4^+/+Apoe^−/− and St3Gal4^−/−Apoe^−/− mice after 12 wk of high-fat diet. HE indicates hematoxylin and eosin. Graphs represent mean±SD; n=6 to 13; Mann–Whitney test. ***P<0.001.

Figure 4. St3Gal4^−/−Apoe^−/− lesions contain fewer leukocytes and display an initial plaque phenotype

A and B, Absolute number of macrophages (Mac2⁺), neutrophils (Ly6G⁺; A), and smooth muscle cells (smoothelin⁺; B) in the aortic root lesions of St3Gal4^+/+Apoe^−/− and St3Gal4^−/−A poe^−/− mice. Representative figures are shown. C, Lesion staging in arches (top) and roots (bottom) according to Virmani et al. D, Necrotic core area (left) and absolute number of apoptotic (TUNEL⁺) cells in the root lesions (right). A to D, Graphs represent mean±SD; n=5 to 10; Mann–Whitney or t test as appropriate. **P<0.01; ***P<0.001.