Deficiency of the Sialyltransferase St3Gal4 Reduces Ccl5-Mediated Myeloid Cell
Recruitment and Arrest
Yvonne Döring1, Heidi Noels2, Manuela Mandl1, Birgit Kramp1, Carlos Neideck1,Dirk Lievens1, Maik
Drechsler1, Remco T. A. Megens1,7, Pathricia V. Tilstam2, Marcella Langer1, Helene Hartwig1, Wendy
Theelen2, Jamey D. Marth3, Markus Sperandio4,5, Oliver Soehnlein1,5,6 and Christian Weber1,5,7
1
IPEK, Ludwig-Maximilians-University, Munich, Germany;2IMCAR, RWTH Aachen University,
Aachen, Germany; 3Center for Nanomedicine, Sanford-Burnham Medical Research Institute, University
of California Santa Barbara, USA; 4WBex, Ludwig-Maximilians-University, Munich, Germany; 5DZHK
(German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Germany; 6AMC,
Amsterdam, the Netherlands, and; 7Cardiovascular Research Institute Maastricht, Maastricht, the
Netherlands.
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Y.D. and H.N. contributed equally to this study.
Running title: St3Gal4-Deficiency Reduces Atherosclerosis
Subject codes:
[134] Pathophysiology
[147] Growth factors/cytokines
[145] Genetically altered mice
Address correspondence to:
Dr. Yvonne Döring
Institute for Cardiovascular Prevention (IPEK)
Pettenkoferstraße 9
80336 München
Tel: 0049-(0)89-5160-4370
([email protected])
Dr. Heidi Noels
Institute for Molecular Cardiovascular Research
(IMCAR)
RWTH Aachen University
Pauwelsstrasse 30
52074 Aachen
Tel : 0049-(0)241-80-37147
[email protected]
In December 2013, the average time from submission to first decision for all original research papers
submitted to Circulation Research was 11.66 days.
DOI: 10.1161/CIRCRESAHA.114.302426
1
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: As the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of
neutrophils and monocytes, we here investigated the role of the sialyltransferase ST3Gal-IV in Ccl5- and
Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis.
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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 Tnf-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:
ST3Gal-IV, leukocyte adhesion, Ccl5, Ccl2, atherosclerosis, chemotine
Nonstandard Abbreviations and Acronyms:
ST3Gal-IV (gene name: St3gal4)
α2-3 sialyltransferase ST3Gal-IV
TNF
tumour necrosis factor
SVEC
SV40-transformed mouse endothelial cell line
DOI: 10.1161/CIRCRESAHA.114.302426
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INTRODUCTION
Glycosyltransferases are involved in the generation of functional selectin ligands, mediating
leukocyte rolling on inflamed endothelium1. Mice deficient of the α2-3 sialyltransferase IV (St3Gal4)
displayed a partially impaired E-selectin ligand function and an almost complete lack of L-selectindependent leukocyte rolling on TNF-α-exposed cremaster muscle venules2-4. More recently, also
leukocyte arrest by the chemokine receptor Cxcr2 was shown to depend on ST3Gal-IV-mediated
sialylation, as St3Gal4-/- mice displayed decreased leukocyte adhesion to inflamed microvessels upon
stimulation with the Cxcr2 ligands Cxcl1 or Cxcl85. In line, human CCR5 requires the attachment of
sialic acid-carrying O-glycans for binding of its chemokine ligands CCL3 and CCL4 and subsequent
receptor activation, as shown by a combination of sialidase treatment and CCR5 mutants in which the
putative binding sites for sialylated O-glycans were exchanged6. Since the chemokine receptors Ccr1 and
Ccr5 (with high-affinity ligand Ccl5) and Ccr2 (binding Ccl2) were previously shown to be important in
myeloid cell recruitment during inflammation7-9, we investigated the role of ST3Gal-IV in Ccl5- and
Ccl2-mediated leukocyte arrest on inflamed endothelium and its effect on atherosclerosis using St3Gal4deficient mice.
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METHODS
Detailed Methods section are provided in the Online Supplement.
Throughout the manuscript, the letter format of all gene and protein notations was chosen to conform with
internationally agreed gene/protein nomenclature guidelines: all letters of human genes/proteins are in
uppercase, whereas for mouse genes/proteins, only the first letter is in uppercase and the remaining letters
are in lowercase. Gene names are in italics.
Atherosclerosis study.
St3Gal4-/- mice10 were crossed with Apoe-/- mice and received a high-fat diet for 12 weeks. Size and
cellular composition of atherosclerotic lesions were assessed by histology and immunofluorescence.
Study of monocytes and neutrophils.
Primary monocytes and neutrophils were isolated from bone marrow with specific cell separation kits
according to the manufacturer’s protocol and were used for chemokine binding assays, flow chamber
adhesion experiments and ex vivo perfusion of mouse carotid arteries. Integrin activation assays and
chemokine binding assays were performed using whole blood, and neutrophils and monocytes were
distinguished using specific fluorescent labeling and flow cytometry.
RESULTS
St3Gal4-/- monocytes and neutrophils show a reduced integrin activation and flow arrest upon Ccl5
stimulation.
Integrin activation is crucial for leukocyte arrest, enabling an efficient interaction with integrin
ligands exposed on the endothelium. Interestingly, Ccl5-induced binding of the integrin ligands Icam1
and Vcam1 was significantly reduced in St3Gal4-/- classical monocytes and neutrophils (Figure 1AB),
which was associated with a significantly decreased ability of Ccl5 to trigger the arrest of St3Gal4-/monocytes and neutrophils on Tnf-α-activated SVECs under flow (Figure 1C). Also, St3Gal4-deficient
DOI: 10.1161/CIRCRESAHA.114.302426
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mice showed a significant decrease in the accumulation of monocytes and neutrophils in the peritoneal
cavity 4 h after intraperitoneal injection of Ccl5 (Figure 1D). Binding of Ccl5 to St3Gal4-/- myeloid cells
was reduced with 30-40% (Online Figure IA), despite comparable surface expression of the high-affinity
Ccl5 receptors Ccr1 and Ccr5, and of the intermediate-affinity receptor Ccr3 (Online Figure II).
Furthermore, enzymatic removal of sialic acids using sialidase treatment decreased Ccl5 binding to
monocytes and neutrophils with 55-80% (Online Figure IB), and seemed associated with reduced
interaction of Ccl5 with both Ccr5 and Ccr1 as shown by sialidase treatment of Ccr1-/- and Ccr5-/myeloid cells, respectively (Online Figure III). Together, these data indicate that sialylation by ST3GalIV and probably other sialyltransferases improve Ccl5 binding, with ST3Gal-IV-mediated sialylation
enabling efficient Ccl5-induced integrin activation and myeloid cell arrest.
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In contrast, St3Gal4-deficiency did not affect Ccl2-induced integrin activation or flow arrest of
neutrophils, and could only significantly reduce binding of Vcam1 but not Icam1 to Ccl2-triggered
monocytes. Correspondingly, Ccl2-induced arrest of St3Gal4-/- monocytes was only marginally reduced
compared to wild-type monocytes, which showed a significantly increased binding upon Ccl2 treatment
(Figure 1AB, Online Figure IVA). Also, no differences were observed in Ccl2 binding to St3Gal4-/- vs
St3Gal4+/+ myeloid cells, or in expression of the Ccl2 chemokine receptor Ccr2 (Online Figure IVB,
Online Figure II). Thus, these data imply that not all chemokines are equally influenced by ST3Gal-IV.
To unravel whether also endothelial ST3Gal-IV affects Ccl5-induced myeloid cell arrest, we
performed ex vivo perfusion assays with mounted and pressurized Tnf-α-activated carotid arteries from
St3Gal4+/+ and St3Gal4-/- mice. Pretreatment of wild-type leukocytes with Ccl5 prior to perfusion
increased their adhesion on endothelium of both wild-type as St3Gal4-/- arteries (Figure 2AB, left panels).
In contrast, Ccl5 pretreatment of St3Gal4-/- leukocytes did not enhance their arrest on either St3Gal4+/+ or
St3Gal4-/- carotid arteries (Figure 2AB, right panels), indicating that ST3Gal-IV on leukocytes but not
endothelial cells enables Ccl5-triggered leukocyte arrest on inflamed endothelium. A role for ST3Gal-IV
in inflammatory cell arrest was further confirmed in vivo by use of intravital fluorescence microscopy of
the carotid artery. St3Gal4-/- mice showed a dramatic reduction in adherent rhodamine 6G-labeled
leukocytes (Online Figure V), despite comparable white blood cell counts (4.3±2.3 (St3Gal4+/+) vs
3.5±2.2 (St3Gal4-/-) x103 leukocytes/µl blood). A decreased arrest was observed for both neutrophils
(CD11b+, Ly6G+) and classical monocytes (CD11b+, Ly6C+) based on i.v. labeling with antibodies to
CD11b, Ly6G and Ly6C (Figure 2CD).
St3Gal4-deficiency reduces atherosclerotic lesion size and myeloid cell influx in mice.
As continuous leukocyte adhesion and influx drive atherosclerotic lesion development11, we
examined a potential role of ST3Gal-IV in atherosclerosis using St3Gal4-/-Apoe-/- and St3Gal4+/+Apoe-/mice on high-fat diet for 12 weeks. Despite comparable leukocyte subpopulations and only small
differences in lipid levels between knock-out and wild-type mice (Online Table), aortic arches, roots and
thoraco-abdominal aortas of St3Gal4-/-Apoe-/- mice displayed a dramatic reduction in plaque development
(Figure 3AB; Online Figure VI). Macrophage and neutrophil numbers in St3Gal4-/-Apoe-/- aortic root
lesions were significantly reduced (Figure 4A), whereas the number of smooth muscle cells was not
altered (Figure 4B). Furthermore, plaque staging of aortic arches and roots according to Virmani et al.12
displayed a very initial lesion phenotype in St3Gal4-/-Apoe-/- mice, characterized by 90% initial xanthomas
in arches and 70% initial xanthomas and pathological intima thickenings in roots of these mice. In
contrast, plaques in wild-type mice showed a very advanced lesion type, mainly represented by fibrous
cap atheromas and fibrocalcification in both arches and roots (Figure 4C). In line, the necrotic core area
and accumulation of TUNEL+ cells was significantly diminished in atherosclerotic plaques of St3Gal4-/Apoe-/- mice (Figure 4D).
Interestingly, immunofluorescent stainings of initial vs advanced human lesions revealed
increased CCL5 levels in advanced compared to initial plaques (Online Figure VII). In line, the smaller
DOI: 10.1161/CIRCRESAHA.114.302426
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root lesions of St3Gal4-/-Apoe-/- mice displayed reduced Ccl5 staining compared to controls (Online
Figure VIII), although Ccl5 serum levels were not significantly changed (Online Figure IX).
DISCUSSION
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In conclusion, this study reveals that the sialyltransferase ST3Gal-IV enables Ccl5-triggered
arrest of monocytes and neutrophils on inflamed endothelium. St3Gal4-/- leukocytes showed a significant
reduction in Ccl5 binding and Ccl5-induced integrin activation despite comparable expression of Ccl5
chemokine receptors. This suggests that sialylation facilitates efficient Ccl5 binding through favourable
conformational changes in Ccl5 receptors, or through enforced electrostatic interactions of basic
chemokine residues with negatively charged sialic acids attached to chemokine receptors6. The
contribution of sialylation to efficient Ccl5 binding is supported by the reduction in Ccl5 binding upon
sialidase treatment, which we observed for both Ccr1-/- and Ccr5-/- leukocytes, and by a previous report
demonstrating human CCR5 to require N-terminal sialylation for efficient chemokine binding6.
Comparably, St3Gal4-deficiency in leukocytes was previously shown to reduce Cxcl8 binding to Cxcr2
and to impair Cxcl1/Cxcr2-triggered neutrophil arrest5. Nonetheless, ST3Gal-IV-mediated sialylation
does not seem a general requirement for efficient chemokine functioning, as Ccl2-triggered leukocyte
arrest was not significantly affected by St3Gal4-deficiency.
Circulating monocytes and neutrophils adhere to and accumulate in atherosclerotic vessels, where
they crucially contribute to atherogenesis11. Recruitment of classical monocytes into atherosclerotic
lesions requires Ccr19 and Ccr57, 9, whereas the precise role of Ccr27, 9, 13 and Cx3cr17, 9 in monocyte
incorporation into lesions has recently been debated. The observed reduction in lesion size in Cx3cr1-/Apoe-/- mice14-16 may rather depend on the role of Cx3cr1 in monocyte and macrophage survival16, instead
of on a direct role of Cx3cr1 in monocyte recruitment into atherosclerotic plaques9. Similarly, although
the reduced atherosclerotic lesion size in Ccr2-/-Apoe-/- mice14, 17 and Ccr2-/- bone marrow chimeras18
clearly indicates an important role for Ccr2 in atherosclerosis, the specific role of Ccr2 in the
incorporation of circulating monocytes into atherosclerotic arteries requires further investigation.
Contradictory findings have been reported on such direct involvement of Ccr2 in lesional monocyte
accumulation9, 13 and suggested that the pro-atherogenic role of Ccr2 may rather be related to a crucial
role for the Ccl2/Ccr2 axis in the mobilization of monocytes from the bone marrow in inflammatory and
atherosclerotic conditions9, 15, 19-22. Compared to monocytes, neutrophils infiltrate atherosclerotic arteries
primarily through Cxcr2, Ccr1, Ccr2 and Ccr58. Thus, the importance of ST3Gal-IV in mediating myeloid
cell arrest in response to Ccl5 and Cxcl1/Cxcl85 - representing high affinity ligands for Ccr1 and Ccr5,
and for Cxcr2, respectively -, could explain why St3Gal4-deficient mice display a severely reduced
leukocyte arrest on inflamed endothelium and an associated decrease in accumulating macrophages and
neutrophils in atherosclerotic vessels. The previous finding that blocking only Ccl5 reduces the arrest of
perfused monocytes on atherosclerotic endothelium with already ~50%23 further supports the importance
of ST3Gal-IV in atherogenic myeloid cell accumulation. Furthermore, the requirement of leukocytic
ST3Gal-IV for the generation of functional selectin ligands2-4 may additionally contribute to reduced
leukocyte rolling and arrest in St3Gal4-/- mice.
Interestingly, atherosclerotic lesion size was previously shown to be strongly correlated with the
number of circulating monocytes, displaying ~90% reduction in atherosclerosis when circulating
monocyte numbers were reduced with comparable extent22. Thus, arrest and infiltration of circulating
monocytes are crucial in determining lesion size, implying that the severely reduced leukocyte arrest in
St3Gal4-/- mice could explain to a great extent their drastic reduction in atherosclerosis. In addition, the
less advanced plaque phenotype combined with a lower platelet count upon St3Gal4-deficiency10 could
underlie the decreased Ccl5 levels in atherosclerotic vessels of St3Gal4-/-Apoe-/- mice, which may then
further add to the reduction in leukocyte recruitment and atherosclerosis progression. Also, it is possible
DOI: 10.1161/CIRCRESAHA.114.302426
5
that reduced Ccl5-induced activation of myeloid cells, as displayed by decreased integrin activation,
further contributes to reduced atherogenesis in St3Gal4-/-Apoe-/- mice. However, further studies are
required to pinpoint the exact role of Ccl5 in atherogenic functions of monocytes and neutrophils, before
being able to address the effect of ST3Gal-IV in this context. In addition, the role of endothelial St3Gal4
in inflammation remains unclear. Although our in vitro data revealed a comparable Ccl5-triggered
leukocyte adhesion to St3Gal4-/- vs St3Gal+/+ carotids after 4 h of Tnf-α stimulation, further studies are
required to address the specific role of St3Gal4 in endothelial activation and in leukocyte adhesion to
chronically inflamed endothelium in more detail in vivo. It is not excluded that St3Gal4-deficiency in
vascular cells further contributes to the drastic reduction in atherosclerosis observed in this study.
Altogether, our data point towards an important contribution of ST3Gal-IV in efficient leukocyte
recruitment and arrest under inflammatory conditions. Hence, targeting sialylation in atherosclerosis, e.g.
by specific inhibitors of ST3Gal-IV, might be a new promising therapeutic approach.
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ACKNOWLEDGMENTS
We thank Yvonne Jansen, Patricia Lemnitzer, Susanne Bierschenk, Melanie Garbe, Stephanie Elbin and
Leon Decker for excellent technical assistance.
SOURCES OF FUNDING
This work was supported by the European Research Council (ERC AdG 249929 to C.W.), the
Netherlands Organisation for Scientific Research (NWO; VIDI project 91712303 to O.S.), the German
Research Foundation (DFG; SO876/3-1, SO876/6-1, FOR809, SFB914-B08 to O.S. and C.W.; SFB914B01 to M.S.), the Else Kröner Fresenius Stiftung (to O.S.), the Mizutani Foundation (090063/2009 to
M.S.) and the LMUexcellent initiative.
DISCLOSURES
C.W. is share holder of Carolus Therapeutics Inc., a company developing chemokine-based antiinflammatory strategies.
REFERENCES
1.
Sperandio M, Gleissner CA, Ley K. Glycosylation in immune cell trafficking. Immunol Rev.
2009;230:97-113
2.
Ellies LG, Sperandio M, Underhill GH, Yousif J, Smith M, Priatel JJ, Kansas GS, Ley K, Marth
JD. Sialyltransferase specificity in selectin ligand formation. Blood. 2002;100:3618-3625
3.
Sperandio M, Frommhold D, Babushkina I, Ellies LG, Olson TS, Smith ML, Fritzsching B, Pauly
E, Smith DF, Nobiling R, Linderkamp O, Marth JD, Ley K. Alpha 2,3-sialyltransferase-iv is
essential for l-selectin ligand function in inflammation. European journal of immunology.
2006;36:3207-3215
4.
Yang WH, Nussbaum C, Grewal PK, Marth JD, Sperandio M. Coordinated roles of st3gal-vi and
st3gal-iv sialyltransferases in the synthesis of selectin ligands. Blood. 2012;120:1015-1026
5.
Frommhold D, Ludwig A, Bixel MG, Zarbock A, Babushkina I, Weissinger M, Cauwenberghs S,
Ellies LG, Marth JD, Beck-Sickinger AG, Sixt M, Lange-Sperandio B, Zernecke A, Brandt E,
Weber C, Vestweber D, Ley K, Sperandio M. Sialyltransferase st3gal-iv controls cxcr2-mediated
firm leukocyte arrest during inflammation. The Journal of experimental medicine.
2008;205:1435-1446
DOI: 10.1161/CIRCRESAHA.114.302426
6
6.
7.
8.
9.
10.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Bannert N, Craig S, Farzan M, Sogah D, Santo NV, Choe H, Sodroski J. Sialylated o-glycans and
sulfated tyrosines in the nh2-terminal domain of cc chemokine receptor 5 contribute to high
affinity binding of chemokines. The Journal of experimental medicine. 2001;194:1661-1673
Tacke F, Alvarez D, Kaplan TJ, Jakubzick C, Spanbroek R, Llodra J, Garin A, Liu J, Mack M,
van Rooijen N, Lira SA, Habenicht AJ, Randolph GJ. Monocyte subsets differentially employ
ccr2, ccr5, and cx3cr1 to accumulate within atherosclerotic plaques. J Clin Invest. 2007;117:185194
Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O. Hyperlipidemia-triggered
neutrophilia promotes early atherosclerosis. Circulation. 2010;122:1837-1845
Soehnlein O, Drechsler M, Doring Y, Lievens D, Hartwig H, Kemmerich K, Ortega-Gomez A,
Mandl M, Vijayan S, Projahn D, Garlichs CD, Koenen RR, Hristov M, Lutgens E, Zernecke A,
Weber C. Distinct functions of chemokine receptor axes in the atherogenic mobilization and
recruitment of classical monocytes. EMBO Mol Med. 2013;5:471-481
Ellies LG, Ditto D, Levy GG, Wahrenbrock M, Ginsburg D, Varki A, Le DT, Marth JD.
Sialyltransferase st3gal-iv operates as a dominant modifier of hemostasis by concealing
asialoglycoprotein receptor ligands. Proceedings of the National Academy of Sciences of the
United States of America. 2002;99:10042-10047
Weber C, Noels H. Atherosclerosis: Current pathogenesis and therapeutic options. Nat Med.
2011;17:1410-1422
Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death:
A comprehensive morphological classification scheme for atherosclerotic lesions.
Arteriosclerosis, thrombosis, and vascular biology. 2000;20:1262-1275
Swirski FK, Libby P, Aikawa E, Alcaide P, Luscinskas FW, Weissleder R, Pittet MJ. Ly-6chi
monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages
in atheromata. J Clin Invest. 2007;117:195-205
Veillard NR, Steffens S, Pelli G, Lu B, Kwak BR, Gerard C, Charo IF, Mach F. Differential
influence of chemokine receptors ccr2 and cxcr3 in development of atherosclerosis in vivo.
Circulation. 2005;112:870-878
Saederup N, Chan L, Lira SA, Charo IF. Fractalkine deficiency markedly reduces macrophage
accumulation and atherosclerotic lesion formation in ccr2-/- mice: Evidence for independent
chemokine functions in atherogenesis. Circulation. 2008;117:1642-1648
Landsman L, Bar-On L, Zernecke A, Kim KW, Krauthgamer R, Shagdarsuren E, Lira SA,
Weissman IL, Weber C, Jung S. Cx3cr1 is required for monocyte homeostasis and atherogenesis
by promoting cell survival. Blood. 2009;113:963-972
Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in ccr2-/- mice reveals a
role for chemokines in the initiation of atherosclerosis. Nature. 1998;394:894-897
Guo J, Van Eck M, Twisk J, Maeda N, Benson GM, Groot PH, Van Berkel TJ. Transplantation of
monocyte cc-chemokine receptor 2-deficient bone marrow into apoe3-leiden mice inhibits
atherogenesis. Arteriosclerosis, thrombosis, and vascular biology. 2003;23:447-453
Serbina NV, Pamer EG. Monocyte emigration from bone marrow during bacterial infection
requires signals mediated by chemokine receptor ccr2. Nat Immunol. 2006;7:311-317
Engel DR, Maurer J, Tittel AP, Weisheit C, Cavlar T, Schumak B, Limmer A, van Rooijen N,
Trautwein C, Tacke F, Kurts C. Ccr2 mediates homeostatic and inflammatory release of gr1(high)
monocytes from the bone marrow, but is dispensable for bladder infiltration in bacterial urinary
tract infection. J Immunol. 2008;181:5579-5586
Tsou CL, Peters W, Si Y, Slaymaker S, Aslanian AM, Weisberg SP, Mack M, Charo IF. Critical
roles for ccr2 and mcp-3 in monocyte mobilization from bone marrow and recruitment to
inflammatory sites. J Clin Invest. 2007;117:902-909
Combadiere C, Potteaux S, Rodero M, Simon T, Pezard A, Esposito B, Merval R, Proudfoot A,
Tedgui A, Mallat Z. Combined inhibition of ccl2, cx3cr1, and ccr5 abrogates ly6c(hi) and
DOI: 10.1161/CIRCRESAHA.114.302426
7
23.
ly6c(lo) monocytosis and almost abolishes atherosclerosis in hypercholesterolemic mice.
Circulation. 2008;117:1649-1657
von Hundelshausen P, Weber KS, Huo Y, Proudfoot AE, Nelson PJ, Ley K, Weber C. Rantes
deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium.
Circulation. 2001;103:1772-1777
FIGURE LEGENDS
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Figure 1. St3Gal4-deficient leukocytes display reduced Ccl5-induced integrin activation and flow
arrest. A-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 binding of Icam1 and Vcam1. n=56. C, Adhesion of perfused monocytes (left) and neutrophils (right) on Tnf-α-activated (10 ng/ml, 4 h)
SVECs after pre-treatment of leukocytes or SVECs with Ccl5 (2 µg/ml, 10 min), as indicated. n=6-12. D,
Intraperitoneal recruitment of monocytes (left) and neutrophils (right) 4 h after i.p. injection of 5 µg Ccl5.
n=11-16. A-D, Graphs represent means ± SD; Mann-Whitney test (A,B) or 1-way ANOVA with Tukey's
multiple comparison test (C,D); *P<0.05, **P<0.01, ***P<0.001.
Figure 2. Reduced Ccl5-induced adhesion of St3Gal4-/- leukocytes on mouse carotid arteries. A-B,
Ex vivo flow adhesion of Ccl5-pretreated (2.5 µg/ml, 10 min) leukocytes on Tnf-α-stimulated (20 ng/ml, 4
h) carotids from St3Gal4+/+ (A) and St3Gal4-/- (B) mice. n=5-7; Mann-Whitney test. C-D, Intravital
microscopy of leukocyte adhesion to Tnf-α-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=6-14; t-test. A-D, Graphs represent means ± 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 weeks of highfat diet. Graphs represent means ± SD; n=6-13; Mann-Whitney test; ***P<0.001.
Figure 4. St3Gal4-/-Apoe-/- lesions contain fewer leukocytes and display an initial plaque phenotype.
A-B, Absolute number of macrophages (Mac2+), neutrophils (Ly6G+) (A) and smooth muscle cells
(smoothelin+) (B) in aortic root lesions of St3Gal4+/+Apoe-/- and St3Gal4-/-Apoe-/- mice. Representative
figures are shown. C, Lesion staging in arches (top) and roots (bottom) according to Virmani et al.12. D,
Necrotic core area (left) and the absolute number of apoptotic (TUNEL+) cells in root lesions (right). AD, Graphs represent means ± SD; n=5-10; Mann-Whitney or t-test as appropriate; **P<0.01,
***P<0.001.
DOI: 10.1161/CIRCRESAHA.114.302426
8
Novelty and Significance
What Is Known?

Chemokine receptors and their ligands play a crucial role in the adhesion of leukocytes on the
endothelium during inflammation.

Receptors for the chemokine Ccl5 are important in mediating inflammatory leukocyte arrest,
particularly in the context of atherosclerosis.

The sialyltransferase α2-3 sialyltransferase ST3Gal-IV is known to be involved in Cxcr2mediated leukocyte arrest on the inflamed endothelium, but it remains unknown whether ST3GalIV also affects the binding of other chemokine ligand-receptor pairs.
What New Information Does This Article Contribute?
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
ST3Gal-IV enables efficient binding of Ccl5 to neutrophils and classical monocytes.

ST3Gal-IV mediates Ccl5-triggered integrin activation and leukocyte arrest on the inflamed
endothelium.

St3Gal4-deficiency reduces atherosclerosis in mice, suggesting that prevention or reduction of
sialylation may be a promising therapeutical approach.
A crucial step in the formation of atherosclerotic lesions is the recruitment and adhesion of neutrophils
and monocytes to the inflamed vascular endothelium, driven by the interaction of chemokines with their
corresponding receptors on the leukocyte cell surface. Whereas the chemokine receptors Ccr1 and Ccr5
are important for the atherogenic recruitment of classical monocytes, neutrophil mobilization and
recruitment is mediated through Cxcr2, Ccr1, Ccr2 and Ccr5. Interestingly, sialylation by sialyltransferase
ST3Gal-IV has been shown to be required for Cxcr2-dependent leukocyte arrest and efficient binding of
Cxcl1 and Cxcl8 to Cxcr2. However, it remains unknown whether ST3Gal-IV also affects other
chemokine receptor-ligand interactions. The results of this study suggest that ST3Gal-IV in myeloid cells
enables efficient binding of Ccl5 (a ligand for the chemokine receptors Ccr1 and Ccr5), and mediates
Ccl5-triggered integrin activation and leukocyte arrest on inflamed endothelium. In contrast, St3Gal4deficiency did not significantly affect binding of Ccl2 (a ligand for Ccr2), or Ccl2-induced flow arrest of
myeloid cells, suggesting that ST3Gal-IV-mediated sialylation is not a general requirement for efficient
chemokine functioning. Corresponding with the important role of the Ccl5 chemokine receptors in the
recruitment of both monocytes and neutrophils to atherosclerotic lesions, inflammatory cell accumulation
and atherosclerosis were severely reduced in St3Gal4-/-Apoe-/- mice. These findings reveal a potentially
important role of sialylation in Ccl5-mediated leukocyte recruitment and arrest under chronic
inflammatory conditions, and suggest that targeting sialylation in atherosclerosis, e.g. by specific
inhibitors of ST3Gal-IV, might be a new promising therapeutical approach.
DOI: 10.1161/CIRCRESAHA.114.302426
9
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Deficiency of the Sialyltransferase St3Gal4 Reduces Ccl5-Mediated Myeloid Cell Recruitment
and Arrest
Yvonne Döring, Heidi Noels, Manuela M Mandl, Birgit Kramp, Carlos Neideck, Dirk Lievens, Maik
Drechsler, Remco T Megens, Pathricia V Tilstam, Marcella Langer, Helene Hartwig, Wendy Theelen,
Jamey D Marth, Markus Sperandio, Oliver Soehnlein and Christian Weber
Circ Res. published online January 14, 2014;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2014 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/early/2014/01/14/CIRCRESAHA.114.302426
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2014/01/14/CIRCRESAHA.114.302426.DC1
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Supplemental Material
Detailed Methods
Mice
St3Gal4-deficient mice1, Ccr1-/-Apoe-/- and Ccr5-/-Apoe-/- mice were bred in the local animal facility
and fed a normal chow diet. Experimental mice were sex- and age-matching. Further, St3Gal4-/- mice
were crossed with Apoe-/- mice to generate St3Gal4-/-Apoe-/- double knockout mice. Female Apoe-/- and
St3Gal4-/-Apoe-/- were fed a high-fat diet containing 21% fat and 0.15% cholesterol (Altromin) starting
at 8 weeks of age for 12 weeks. Mouse strains were all on C57Bl/6 background. All animal
experiments were approved by the local ethical committee.
Isolation of primary monocytes and neutrophils
Primary monocytes and neutrophils of St3Gal4+/+ and St3Gal4-/- mice were isolated from the bone
marrow with cell separation kits from Miltenyi Biotec according to the manufacturer’s protocol. For
monocytes we used the ‘CD115 MicroBead Kit’, for neutrophils the ‘Neutrophil Isolation Kit, mouse’.
The isolated cells were used for chemokine binding assays, flow chamber adhesion experiments and ex
vivo perfusion of mouse carotid arteries.
Integrin activation assay
Whole blood (100 µl) obtained from the retro-orbital plexus of St3Gal4+/+ and St3Gal4-/- mice was
subjected to red-blood-cell lysis (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA-Na2, pH 7.4) and
leukocytes were suspended in Hanks Balanced Salt Solution (HBSS) containing 1 mM CaCl2 and
MgCl2 (Invitrogen) and 0.5% BSA (Sigma). Cells were exposed to Ccl2 and Ccl5 (2 µg/ml,
Peprotech) or an equal volume of buffer, in the presence of Icam1/Fc (recombinant mouse Icam1
fused to human IgG1 Fc; 10 μg/ml, R&D Systems) or Vcam1/Fc (10 μg/ml, R&D Systems) and PEconjugated anti-human IgG1 (1 µg/ml; Fc-specific, Southern Biotechnology) for 5 min at 37°C. After
washing, cells were labeled with antibodies to CD45, CD115, Ly6G and Gr1 to identify classical
monocytes and neutrophils. Binding of Icam1 or Vcam1 was measured by flow cytometry.
Ccl5 and Ccl2 binding assay and enzymatic desialylation
Whole blood (100 µl) obtained from the retro-orbital plexus of mice was EDTA-buffered and
subjected to red-blood-cell lysis (Pharmlyse BD Biosciences). Cells were further stained with an
antibody cocktail against CD45 (-APC-Cy7, BD Biosciences, 30-F11), CD115 (-PE, BD Bioscience,
AFS98), Gr1 (-PerCP, eBioscience, RB6-8C5), CD11b (-efluor 450, eBioscience, clone M1/70) and
Ly6G (-FITC, BioLegend, clone 1A8) and washed once. For analysis of Ccl5 binding, 0.5 µg murine
Ccl5 (Peprotech) was added to each sample (5x105 cells) and incubated for 10 min on ice. Cells were
washed again and stained for 10 min on ice with an anti-Ccl5 biotin-strep-PE-Cy7 antibody (Abcam,
ab83135), which had been streptavidin-PE-Cy7 labeled (BD Pharmingen) and washed a priori. Cells
stained with the anti-Ccl5 biotin-strep-PE-Cy7 antibody without Ccl5 incubation served as a negative
control. For analysis of Ccl2 binding, 0.5 µg murine Ccl2 (Peprotech) was added to each sample
(5x105 cells) and incubated for 10 min on ice. Cells were washed and stained for 10 min on ice with an
anti-Ccl2 FITC-labeled antibody. Cells stained with the anti-Ccl2 FITC-labeled antibody without Ccl2
incubation served as a negative control. Binding of Ccl5 and Ccl2 to neutrophils
(CD45+CD11b+CD115-Ly6G+) and classical monocytes (CD45+CD11b+CD115+Ly6G-Gr1high) was
assessed by flow cytometry (FACSCantoII, Beckton Dickenson) and results were analyzed using
FlowJo software (Tree Star). Bar graphs depict mean binding capacity as MFI – MFI negative control,
calculated as % of the wild-type mice.
To induce enzymatic desialylation, 100 µl blood was subjected to red-blood-cell lysis and
resuspended in 100 µl buffer (5 mM Na-acetate, 15.4 mM NaCl, 0.9 mM CaCl2, 3.4 mg/ml BSA, 1.3
mM EDTA in HBSS), containing 100 U/ml purified sialidase (Roche Diagnostics) for 30 min at 37°C.
Subsequently, cells were washed and investigated for chemokine binding capacity.
Parallel flow chamber adhesion assay
Flow chamber adhesion assays were performed as previously described2. Briefly, mouse endothelial
cells (SVECs) grown to confluency in 35-mm petri dishes were stimulated with mouse Tnf-α (10
ng/ml) for 4 h. Thereafter, dishes were assembled as the bottom of a parallel wall flow chamber and
mounted on the stage of an Olympus IMT-2 inverted microscope with 20× and 40× phase contrast
objectives. Monocytes and neutrophils (0.5 × 106/ml) were stained with 0.3 μg/mL calcein AM
(Molecular Probes) in assay buffer (HBSS, 10 mM Hepes pH 7.4, 0.5 % BSA). Cells suspensions
were kept in a heating block at 37 °C during the assays and were perfused into the flow chamber at a
shear rate of 1.5 dyn/cm2 for 5 min. Where indicated, SVEC, monocytes or neutrophils were pretreated
with mouse Ccl5 (2 µg/ml, Peprotech) or Ccl2 (2 µg/ml, Peprotech) for 10 min before the flow
experiment. After 2 min of perfusion, adherent cells were quantified in multiple fields (0.24 mm2 each,
>10 per treatment) by analysis of images recorded with a long integration 3CCD video camera (JVC,
Japan) using AnalySIS software (Soft Imaging System, Münster, Germany).
Intraperitoneal recruitment assay
In vivo peritoneal recruitment of neutrophils and classical monocytes was determined in St3Gal4+/+
and St3Gal4-/- mice, which were injected intraperitoneally (i.p.) with sterile PBS with or without 5 µg
mouse Ccl5. After 4 h, the cell infiltrate was harvested by intraperitoneal lavage with ice-cold HBSS
containing 5 mM EDTA, stained with antibodies against CD45, CD115, Gr1, CD11b or Ly6G and
analyzed by FACS. The absolute number of recruited cells was determined by CountBright absolute
counting beads (Invitrogen). Neutrophils were discriminated being CD11b+Ly6G+, classical
monocytes were determined as CD115+Gr1+ cells.
Ex vivo perfusion of carotid arteries
Carotid artery segments (common part) were carefully excised from 10- to 16-weeks-old St3Gal4+/+
and St3Gal4-/- mice to avoid tissue damage and stored in HBSS at room temperature prior to
examination. For examination with fluorescence microscopy, the arteries were mounted between glass
pipettes in a home-built perfusion chamber3, 4, transmural pressure (80 mm Hg) was controlled by
adaption of the height of the outflow tract to mimic physiological conditions. After prestimulation of
the mounted and pressurized carotids with Tnf-α (20 ng/ml, 4 h) in HBSS, the carotids were perfused
at 100 μl/min with calcein-AM labeled primary neutrophils or monocytes (1x106 cells/ml) in
physiological direction. Where indicated, leukocytes were pretreated with Ccl5 (2.5 µg/ml) for 10 min
before perfusion. After 8 min of perfusion (cells were perfused for 5 min followed by 3 min buffer
perfusion), arrest was analyzed using an upright Olympus BX51 microscope equipped with a
Hamamatsu 9100-02 EMCCD camera and a 10x saline-immersion objective, as previously described5.
For image acquisition and analysis Olympus Cell-R software was used.
Intravital microscopy
Leukocyte adhesion to the carotid artery was analyzed in St3Gal4+/+ and St3Gal4-/- mice via intravital
microscopy, as described previously6. The right jugular vein was canulated with a catheter for
antibody and dye injection. After exposure of the left carotid artery, antibodies (1 µg) to CD11b
(650NC, ebioscience), Ly6G (BioLegend) and Ly6C (ebioscience) were sequentially administered to
label various leukocyte subsets. Recordings were made 3 min after injection of each antibody. Finally,
rhodamine 6G (100 µl of a 0.1‰ solution) was injected to label all circulating leukocytes. Intravital
microscopy was performed using an Olympus BX51 microscope equipped with a Hamamatsu 9100-02
EMCCD camera and a 10x saline-immersion objective. For image acquisition and analysis Olympus
Cell-R software was used. For induction of arterial inflammation, carotid arteries were locally treated
with Tnf-α (100 ng) prior to intravital microscopy.
Lipids and atherosclerotic lesion development
Cholesterol and triglyceride levels in mouse serum were quantified using enzymatic assays (Roche or
BioTrend) according to the manufacturer’s protocol. Leukocyte counts were determined by routine
laboratory assays. The extent of atherosclerotic lesion development was analyzed as previously
described7, 8. Briefly, atherosclerosis was assessed in transverse cryo-sections of aortic roots and en
face prepared aortas by staining for lipid depositions with Oil-red-O. Lesion development in aortic
arches and main branch points (brachiocephalic artery, right and left subclavian artery, and right and
left common carotid artery) was quantified by HE-staining using computerized image analysis (Diskus
Software) and Leica Qwin Imaging software (Leica Ltd.). Aortic root lesions were stained with an
antibody to Ly6G (1A8, BD Biosciences), Mac2 (AbD Serotec), smoothelin (N-15, Santa Cruz) or
Ccl5 (53405, R&D Systems). Nuclei were counter-stained by 4',6-Diamidino-2-phenylindol (DAPI).
After incubation with a secondary FITC-conjugated antibody (Life Technologies) for 30 min at room
temperature, sections were analyzed using a Leica DMLB fluorescence microscope and chargecoupled device (CCD) camera. The absolute number of Ly6G+, Mac2+ and smoothelin+ cells in the
lesions were quantified per aortic root section. Furthermore, TUNEL staining was performed using In
Situ Cell Death Detection Kit, TMR red (Roche) to assess the number of apoptotic/necrotic cells
within aortic root sections. For each mouse and staining, 2-3 root sections were analyzed and
averaged. Examination of carotid artery intimal xanthoma (early lesion) or thin fibrous cap atheroma
(advanced lesion, according to Virmani classification9) from patients undergoing endarterectomy was
approved by an institutional review committee and performed with the patients’ informed consent, in
accordance with institutional guidelines. The human lesions were stained with an antibody against
CCL5 (whole goat IgG, AB-278-NA, R&D Systems) or an appropriate isotype control IgG, and
visualized by a Cy3-conjugated anti-goat IgG antibody (Sigma Aldrich). Cells were counterstained by
DAPI.
Flow cytometry
Whole blood obtained from the retro-orbital plexus of mice was EDTA-buffered and subjected to redblood-cell lysis. Blood leukocytes were discriminated by the following antibody cocktail: anti-CD45,
anti-CD115, anti-Gr1, anti-CD11b, anti-CD19 (ebioscience, clone MB19-1) and anti-CD3
(ebioscience, clone 145-2C11). Leukocyte subsets were defined using FlowJo software: neutrophils
(CD45+CD115-Gr1high), monocytes (CD45+CD115+), classical monocytes (CD45+CD115+Gr1high),
non-classical monocytes (CD45+CD115+Gr1low) and lymphocytes (CD45+CD3+ and CD45+CD19+).
Chemokine receptors have been identified with anti-Ccr1 FITC (R&D, FAB5986F), anti-Ccr3 Alexa
Fluor 647® (BioLegend, clone TG14/Ccr3), anti-Ccr5 PE (ebioscience, clone HM-CCR5) and antiCcr2 PE (R&D Systems).
ELISA
Ccl5 levels in mouse serum were measured using a mouse Ccl5 ELISA kit (R&D Systems), according
to the manufacturer’s protocol.
Statistics
All data are expressed as means ± SD. Statistical calculations were performed using GraphPad Prism 5
(GraphPad Software Inc.). Unpaired Student’s t-test, Mann-Whitney or one-way ANOVA with
Tukey’s Multiple Comparison test were used, as appropriate. P-values < 0.05 were considered as
being statistically significant.
Supplemental Literature
1. Ellies LG, Ditto D, Levy GG, Wahrenbrock M, Ginsburg D, Varki A, Le DT, Marth JD.
Sialyltransferase st3gal-iv operates as a dominant modifier of hemostasis by concealing
asialoglycoprotein receptor ligands. Proceedings of the National Academy of Sciences of the United
States of America. 2002;99:10042-10047
2. Weber KS, von Hundelshausen P, Clark-Lewis I, Weber PC, Weber C. Differential immobilization
and hierarchical involvement of chemokines in monocyte arrest and transmigration on inflamed
endothelium in shear flow. European journal of immunology. 1999;29:700-712
3. Megens RT, Oude Egbrink MG, Cleutjens JP, Kuijpers MJ, Schiffers PH, Merkx M, Slaaf DW,
van Zandvoort MA. Imaging collagen in intact viable healthy and atherosclerotic arteries using
fluorescently labeled cna35 and two-photon laser scanning microscopy. Mol Imaging. 2007;6:247260
4. Schmitt MM, Megens RT, Zernecke A, Bidzhekov K, van den Akker NM, Rademakers T, van
Zandvoort MA, Hackeng TM, Koenen RR, Weber C. Endothelial jam-a guides monocytes into
flow-dependent predilection sites of atherosclerosis. Circulation. 2013
5. Doring Y, Drechsler M, Wantha S, Kemmerich K, Lievens D, Vijayan S, Gallo RL, Weber C,
Soehnlein O. Lack of neutrophil-derived cramp reduces atherosclerosis in mice. Circ Res.
2012;110:1052-1056
6. Soehnlein O, Drechsler M, Doring Y, Lievens D, Hartwig H, Kemmerich K, Ortega-Gomez A,
Mandl M, Vijayan S, Projahn D, Garlichs CD, Koenen RR, Hristov M, Lutgens E, Zernecke A,
Weber C. Distinct functions of chemokine receptor axes in the atherogenic mobilization and
recruitment of classical monocytes. EMBO Mol Med. 2013;5:471-481
7. Lutgens E, Lutgens SP, Faber BC, Heeneman S, Gijbels MM, de Winther MP, Frederik P, van der
Made I, Daugherty A, Sijbers AM, Fisher A, Long CJ, Saftig P, Black D, Daemen MJ, Cleutjens
KB. Disruption of the cathepsin k gene reduces atherosclerosis progression and induces plaque
fibrosis but accelerates macrophage foam cell formation. Circulation. 2006;113:98-107
8. Doring Y, Soehnlein O, Drechsler M, Shagdarsuren E, Chaudhari SM, Meiler S, Hartwig H,
Hristov M, Koenen RR, Hieronymus T, Zenke M, Weber C, Zernecke A. Hematopoietic interferon
regulatory factor 8-deficiency accelerates atherosclerosis in mice. Arteriosclerosis, thrombosis, and
vascular biology. 2012;32:1613-1623
9. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death:
A comprehensive morphological classification scheme for atherosclerotic lesions. Arteriosclerosis,
thrombosis, and vascular biology. 2000;20:1262-1275
Supplemental Table and Figures with Legends
Online Table: Leukocyte subpopulations and lipid profiles of St3Gal4+/+, St3Gal4-/-, Apoe-/St3Gal4+/+ and Apoe-/-St3Gal4-/- mice. Peripheral blood leukocyte subsets were measured by flow
cytometry. All values are displayed as mean ± SD (n=6-14).
Blood cells
as % leukocytes
Neutrophils
St3Gal4
17.5 ± 3.8
St3Gal4
12.9 ± 5.1
Apoe St3Gal4
17.5 ± 10.1
Monocytes
classical
non-classical
7.5 ± 2.5
5.3 ± 2.5
2.3 ± 0.9
10.8 ± 4.1
8.8 ± 1.8
2.8 ± 1.2
9.6 ± 3.8
4.5 ± 1.8
3.1 ±1.6
9.0 ± 4.8
5.3 ± 1.6
3.1 ± 2.5
Lymphocytes
59.8 ± 9.0
62.6 ± 6.2
55.7 ± 9.3
56.2 ± 5.7
Lipids
mg/dl
Cholesterol
St3Gal4
283.6 ± 84.3
St3Gal4
288.1 ± 92.6
Apoe St3Gal4
746.1 ± 62.6
Triglycerides
62.6 ± 15.3
37.6 ± 9.3
50.2 ± 10.9
+/+
+/+
-/-
-/-
-/-
-/-
+/+
+/+
-/-
Apoe St3Gal4
17.7 ± 6.2
-/-
Apoe St3Gal4
610.2 ± 90.5
79.9 ± 8.9
-/-
-/-
Online Figure I. St3Gal4-deficiency and sialidase treatment reduce Ccl5 binding. A, Ccl5 binding
to peripheral blood monocytes and neutrophils from St3Gal4+/+ and St3Gal4-/- mice, represented as
mean fluorescence intensity (MFI) after flow cytrometric detection of a fluorescent anti-Ccl5 antibody.
Bar graphs depict MFI – MFI negative control, calculated as % of the wild-type mice. n=14-16; t-test.
Representative histograms are shown. B, Ccl5 binding to peripheral blood monocytes and neutrophils
from C57Bl/6 mice, without or with sialidase treatment, as indicated. n=5-6; Mann-Whitney test. A-B,
Graphs represent means ± SD. **P<0.01.
Online Figure II. Surface expression of chemokine receptors Ccr1, Ccr3, Ccr5 and Ccr2 on
neutrophils and classical monocytes does not differ between St3Gal4+/+ and St3Gal4-/- mice.
Peripheral blood leukocytes were incubated with an antibody cocktail (to CD115, Ly6G, CD11b, Gr1
and CD45) to characterize neutrophils and classical monocytes and with antibodies against Ccr1,
Ccr3, Ccr5 or Ccr2. Grey lines depict FMO controls. Fluorescence intensity was measured by FACS.
One representative histogram for each experiment is shown (n=3).
Online Figure III. Sialidase treatment reduces Ccl5 binding to both Ccr1-/- and Ccr5-/- myeloid
cells. A-B, Ccl5 binding to peripheral blood monocytes and neutrophils from Ccr1-/-Apoe-/- mice (A)
and Ccr5-/-Apoe-/- mice (B), without or with sialidase treatment, as indicated. Ccl5 binding is
represented as mean fluorescence intensity (MFI) after flow cytrometric detection of a fluorescent
anti-Ccl5 antibody. Bar graphs depict MFI – MFI negative control, calculated as % of the untreated
cells. Graphs represent means ± SD; n=4-5; Mann-Whitney test; *P<0.05.
Online Figure IV. St3Gal4-deficiency does not significantly reduce Ccl2-induced flow arrest of
Ccl2 binding. A, Adhesion of perfused classical monocytes and neutrophils on Tnf-α-activated (10
ng/ml, 4 h) SVECs after pre-treatment of leukocytes or SVECs with Ccl2 (2 µg/ml, 10 min), as
indicated. n=8-10; 1-way ANOVA with Tukey's multiple comparison test. B, Ccl2 binding to
peripheral blood monocytes and neutrophils from St3Gal4+/+ and St3Gal4-/- mice, represented as MFI
after flow cytrometric detection of a fluorescent anti-Ccl2 antibody. n=15-18. A-B, All graphs
represent means ± SD. **P<0.01.
Online Figure V. Reduced adhesion of leukocytes in St3Gal4-/- mice in vivo. Interactions of
leukocytes with the carotid artery were visualized by intravital microscopy of St3Gal4+/+ and St3Gal4/mice after injection of Rhodamine-6G to label all circulating leukocytes. Bars represent means ± SD;
n=14; t-test; ***P<0.001. Representative pictures are shown at the left.
Online Figure VI. Reduced lesion size in aortas of St3Gal4-/-Apoe-/- mice. En face aortas were
stained with Oil-red-O and analyzed for the extent of lesion development. The graph shows means ±
SD; Mann-Whitney test; *P<0.05. Representative pictures are shown at the left.
Online Figure VII. CCL5 is enriched in advanced human plaque specimens. Early and advanced
human atherosclerotic specimens were stained for CCL5. Representative images for each plaque stage
are depicted.
Online Figure VIII. Reduced Ccl5 levels in the smaller root lesions of St3Gal4-/-Apoe-/- mice.
Shown are representative images of immunofluorescent Ccl5 stainings of root lesions of St3Gal4-/Apoe-/- and St3Gal+/+Apoe-/- mice after 12 weeks of high-fat diet.
Online Figure IX. Systemic Ccl5 serum levels were not altered in St3Gal4-/-Apoe-/- mice
compared to control animals. Ccl5 serum levels in St3Gal4+/+Apoe-/- and St3Gal4-/-Apoe-/- mice after
12 weeks of high-fat diet were measured by ELISA. The graph shows means ± SD; n=8-12.