Supplemental Materials and Methods s2

Schulz et al. CIRCULATIONAHA/2007/695189

Supplemental Materials and Methods

Antibodies and Reagents

TNF- and IFN- were from PeproTech (London W68LL, UK). Recombinant human fractalkine (rhCX3CL1) and anti-human fractalkine (CX3CL1) antibodies (mAb365, clone

51637.11, and goat polyclonal anti-human antibody AF365) were from R&D Diagnostics

(Minneapolis, MN). The anti-human fractalkine receptor (CX3CR1) antibody (TP502) was purchased from Torrey Pines Biolabs (Houston, TX). Further, we used mouse antibodies recognizing human CD11b, CD14, CD41, CD54, CD62P (Beckman Coulter, Krefeld,

Germany), CD45 (DakoCytomation, Hamburg, Germany), CD62E (Calbiochem, Schwalbach,

Germany) and CD106 (Serotec, Düsseldorf, Germany). FITC-labeled secondary rabbit anti- mouse antibody (F0261) was from DakoCytomation. Function blocking antibodies were

1 directed against human αIIbβ3 integrin (c7E3, Lilly, Bad Homburg, Germany) , GPIbα (SZ2,

Beckman Coulter) or CD62P (AK-4, Becton Dickinson Biosciences). We obtained goat polyclonal control IgG (Jackson ImmunoResearch, Dianova, Hamburg, Germany) as well as mouse IgG1 isotype controls (Becton Dickinson Biosciences, Heidelberg, Germany).

Assessment of leukocyte adhesion to atherosclerotic carotid arteries in ApoE-deficient

mice in vivo

Intravital microscopic studies of murine carotid arteries were performed as previously described2,3. ApoE-deficient mice on C57BL/6J background were purchased from Charles

River Laboratories. At the age of 4-weeks mice were fed a western type high-cholesterol diet for another 14 weeks, experiments were performed using 18 weeks old male animals.

The mouse monocyte cell line (WEHI 274.1) was purchased from European Collection of

Cell Cultures (Porton Down, Salisbury, Wiltshire, UK) and maintained in suspension culture as previously described4. For experiments WEHI 274.1 were labeled with 5- carboxyfluorescein diacetate succinimidyl ester (DCF) and 5x106 cells were injected

1 Schulz et al. CIRCULATIONAHA/2007/695189 intravenously into the jugular vein of anesthetized ApoE-/- mice3. Interaction of leukocytes with the vascular wall of atherosclerotic carotids was visualized using a BX51W1 microscope

(20x water immersion objective, Olympus) equipped with a MT20 monochromator for epi- illumination as reported previously2,3. To study the role of fractalkine for leukocyte recruitment in vivo, we pre-treated mice with either a function blocking rabbit anti-mouse

CX3CL1 polyclonal antibody (TP233, Torrey Pines Biolabs, Houston, TX) or rabbit IgG.

TP233 or control IgG were pre-injected 24hrs (100 µg intraperitoneal) and 15min (50 µg intravenous) prior to experiments5. The number of adherent leukocytes was quantified 15 minutes after injection of WEHI cells and given as n/mm2 surface area (n=4 carotid arteries per group).

Generation of Flp-In CHO cells stably expressing membrane-bound human fractalkine

The Flp-In system (Invitrogen, Carlsbad, CA) was used to establish a Flp-In CHO cell line stably expressing membrane-bound full-length fractalkine. In brief, the coding sequence of human fractalkine (GenBank accession number BC001163) was amplified from the mammalian expression vector pcDNA3 containing full length fractalkine cDNA (kindly provided by D. R. Greaves, Sir William Dunn School of Pathology, University of Oxford,

UK) using the primers 5`-GCGGCCGCTAGCGCCACCATGGCTCCGATATCTCTGTCG-

3` and 5`-GCGGCCGGATCCTCACACGGGCACCAGGACATA-3`. The resultant PCR fragment was cloned into the NheI and BamHI restriction sites of the expression vector pcDNA5-FRT (Invitrogen) to obtain the plasmid pcDNA5-human fractalkine. Flp-In CHO host cells (Invitrogen) were cotransfected with a 9:1 ratio (w/w) of the plasmids pOG44:pcDNA5-hFKN. After 48 h, transfected cells were selected in medium containing 500

µg/mL hygromycin B. Hygromycin-resistant foci were picked approximately 2 weeks post- transfection, expanded and split into duplicates, with one set analyzed for cell surface expression of human fractalkine by fluorescence-activated cell sorting (antibodies: mouse

2 Schulz et al. CIRCULATIONAHA/2007/695189 anti-human fractalkine clone 51637.11, and secondary FITC-labeled anti-mouse antibody).

TM Highly hCX3CL1-positive recombinant clones were sorted using a MoFlo high performance cell sorter (DakoCytomation) achieving a purity of >98%. Thereafter, transfected cells were cloned by limited dilution and subsequently maintained in the presence of 500 µg/mL hygromycin. Nontransfected Flp-In CHO host cells and pcDNA5/FRT mock transfected Flp-

In CHO cells served as controls.

Preparation of leukocytes, platelets and platelet-depleted whole blood

Human whole blood from healthy volunteer donors, who had not taken any medication for at least 10 days before the experiment, was collected into syringes containing 0.5 vol% heparin.

Where indicated, whole blood was depleted from platelets by magnetic bead separation using a CD41-antibody, thus inducing a reduction in platelet count of > 99.6% (residual platelet count < 1,000 platelets per µl blood).

Washed human platelets were isolated from whole blood as described1 (final concentration

2x108/mL) and resuspended into HEPES-modified Tyrode solution (mmol/L: NaCl, 140;

HEPES, 5; D-glucose, 5; KCl, 4; MgCl2, 1; CaCl2, 1; NaHCO3, 12; and 1 mg/mL BSA (pH

7.4)).

Leukocytes were sorted from lysed whole blood using a FACS Aria flow cytometer (Becton

Dickinson Biosciences). Leukocytes were identified by analysis of CD45 positive cells, whereas platelet (CD41) positive cells were excluded to avoid isolation of platelet-leukocyte aggregates. Isolated leukocytes were resuspended into buffer achieving a final concentration of 1x105 cells/mL.

Assessment of fractalkine protein expression using Western blot analysis

HUVEC were lysed in pre-chilled RIPA buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L

NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, complete protease inhibitors;

3 Schulz et al. CIRCULATIONAHA/2007/695189

Roche, East Sussex, UK). After 30 min of incubation on ice, cell debris was removed by centrifugation at 16,000 g for 15 min. From each sample 27 µg of total protein were separated under reducing conditions on a 8-16% precast Novex Tris-glycine gradient gel (Invitrogen,

Karlsruhe, Germany) and transferred to a Hybond nitrocellulose membrane (Amersham

Pharmacia Biotech UK, Little Chalfont, Buckingham, UK). The immunoblot was developed according to standard protocol using a polyclonal goat anti-human fractalkine antibody and a secondary horseradish-peroxidase-linked anti-goat IgG (Alexis Deutschland GmbH,

Grünberg, Germany). To verify equal protein loading of each lane, membranes were reprobed with a polyclonal antibody recognizing total actin protein (goat anti-human actin (I-19), 0.2

µg/mL final concentration; Santa Cruz Biotechnology, Santa Cruz, CA).

Assessment of CX3CL1 and adhesion molecule expression by flow cytometry

CX3CL1 expression was analyzed on cytokine-activated or non-stimulated HUVEC that were either cultivated or primarily isolated. In parallel, we also addressed CX3CL1 expression on fractalkine transfected or mock Flp-In CHO cells using the same protocol. HUVEC or CHO cells were incubated with an anti-human CX3CL1 antibody (10 µg/mL) and a FITC-labeled secondary antibody (10 µg/mL) for 15min. Thereafter, cells were washed, detached with trypsin/EDTA, and fixed with paraformaldehyde (0.5%). 20,000 events were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson Biosciences, Heidelberg, Germany).

Cells were gated by their characteristic forward and side scatter distribution8 and the mean intensity of immunofluorescence was used as index of antigen surface expression.

Unstimulated (resting) HUVEC and mock Flp-In CHO cells, respectively, served as controls.

To analyze the expression of adhesion molecules on stimulated endothelium, HUVEC were treated with 50 ng/mL TNF- and 20 ng/mL IFN- for 20hrs and monoclonal FITC- labeled mouse anti-human antibodies directed either against CD106, CD62E, CD54, CD62P,

CD11b or a mouse IgG1 isotype control were added. Flow cytometric analysis was performed as described above.

Determination of platelet activation by soluble and membrane-bound CX3CL1 using

flow cytometry

Surface expression of platelet P-selectin was determined on washed human platelets by flow cytometry using an anti-human CD62P antibody9. To address the effect of fractalkine on platelet degranulation (CD62P expression) platelets were incubated with soluble full- length recombinant human fractalkine (rhCX3C; final concentration 1 µg/mL) for 15min in the absence or presence of 1 µmol/L ADP.

To determine the effects of surface adherent fractalkine on platelet degranulation, 24- well plates were coated with laminin (5µg/mL) or rhCX3C (1 or 5µg/mL) and blocked overnight with 2% BSA. Washed human platelets were added to the coated 24-well plates and co-incubated for 60min under cell culture conditions. Thereafter, all platelets were removed by gentle washing and CD62P immunoreactivity was quantified by flow cytometric analysis.

To additionally identify the effects of membrane-bound fractalkine, washed platelets were perfused over fractalkine-transfected or mock CHO cells, respectively, for 10min at a shear rate of 1000/s using the flow chamber system described below. Perfused CHO cells were washed by perfusion of HEPES-modified Tyrode solution for additional 5min at the same shear rate. Adherent platelets were directly co-stained in the flow chamber with anti- human CD62P-FITC and CD41-PE antibodies. After labeling, cells were washed gently, detached with trypsin/EDTA, and fixed with paraformaldehyde (0.5%). CHO cells carrying platelets were gated by detection of CD41-PE, and CD62P expression was quantified from

100 adherent platelets.

To determine the effect of fractalkine on platelet-leukocyte interactions, whole blood was incubated with soluble full-length fractalkine (rhCX3C; final concentration 1 µg/mL) and

5 Schulz et al. CIRCULATIONAHA/2007/695189 leukocyte-platelet co-aggregation was assessed as previously described10. In brief, platelet

(CD41) positive cells were identified within the leukocyte population (CD11b positive cells) by flow cytometric analysis. Results were given in percent aggregate formation compared to non-stimulating conditions.

Evaluation of leukocyte and platelet adhesion under flow conditions

Experiments were basically performed as previously described1,11. Glass cover slips for usage in a flow chamber were cultivated until confluence was obtained with either HUVEC, which were subsequently stimulated with TNF- and IFN- for 20hrs, or Flp-In CHO cells transfected with human full-length fractalkine or mock, respectively.

To analyze and quantify leukocyte adhesion to activated endothelial cells under arterial flow conditions, leukocytes and platelets were labeled in whole blood by incubation with the fluorescent dye rhodamine-6G (final concentration 0.2 g/L) for 15min at 37°C.

Leukocytes could be readily distinguished from platelets by their larger size and nuclear morphology; red cells were not visualized by rhodamine-6G. Before perfusion, stimulated

HUVEC were incubated with buffer (Dulbecco´s phosphate buffered saline, Sigma,

Deisenhofen, Germany) containing a function-blocking goat anti-human fractalkine (anti-

CX3CL1) antibody or a non-binding, irrelevant goat anti-human IgG control antibody for

30min. Where indicated, whole blood was preincubated with antibodies against either the fractalkine receptor (CX3CR1 antibody), P-selectin or IgG1 isotype control antibody. Anti-

CX3CL1 and anti-CX3CR1 were used at different concentrations (1, 5 and 10 µg/mL) alone or in combination (with anti-CX3CL1 for blocking fractalkine on HUVEC and anti-CX3CR1 for whole blood incubation) as indicated.

Perfusion was performed for 10min at 1000/s equivalent to shear forces found in medium sized arteries under physiological conditions (reviewed in12), followed by a 5min perfusion with HEPES-modified Tyrode solution at a shear rate of 1000/s using a pulse-free

6 Schulz et al. CIRCULATIONAHA/2007/695189 pump. The flow chamber was mounted on an inverted fluorescence microscope (Axiovert,

Zeiss, Jena, Germany) and fluorescent images were recorded from 5-8 different microscopic fields (20x objectives) using a digital photo camera (Axiocam, Zeiss, Jena, Germany). The number of firmly adherent leukocytes was counted from the recorded fluorescent images and is given in percent of the maximum adhesion achieved by TNF- /IFN- co-stimulation

(without addition of antibodies) of each experiment. Confluence and integrity of the endothelial cell layer before and after flow chamber experiments with exposure to high shear forces, was confirmed using confocal microscopy as described. Briefly, HUVEC-coated cover slips were fixed in paraformaldehyde, immunostained with a FITC-labeled antibody against platelet-endothelial-cell adhesion molecule 1 (PECAM-1, CD31) and examined by confocal microscopy13. To further characterize the subtypes of leukocytes that become adherent to activated HUVEC, the cover slips were gently removed from the chamber and differentially stained with May-Grunwald/Giemsa stain as described previously14.

To define the adhesion molecules involved in platelet adhesion to activated endothelium and to define the role of platelet adhesion for leukocyte recruitment, washed platelets or whole blood were labeled with rhodamine-6G and perfused for 10min at 1000/s over TNF- /IFN- co-stimulated HUVEC in the presence of the monoclonal antibody c7E3

(4 µg/mL), an anti-human GPIbα mAb (10 µg/mL), or both mAbs in combination. To address the role of membrane-bound fractalkine for platelet adhesion, stimulated HUVEC were incubated with antibodies directed against fractalkine (anti-CX3CL1) or IgG prior to perfusion.

To address the role of membrane-bound fractalkine and P-selectin for leukocyte and platelet adhesion, whole blood labeled with 5-carboxyfluorescein diacetate succinimidyl ester

(DCF) as described previously3 were perfused for 10min at 1000/s over fractalkine or mock transfected CHO cells in the presence of a neutralizing anti-P-selectin mAb (10 µg/mL) or

7 Schulz et al. CIRCULATIONAHA/2007/695189 mouse IgG1 control. Leukocytes were identified using FITC-conjugated anti-CD45 mAb (20

µg/mL).

To compare the influence of shear stress on leukocyte adhesion under flow, we studied firm adhesion as well as tethering/rolling of leukocytes gained from different preparations

(whole blood, isolated leukocytes resuspended into buffer, presence and absence of platelets) on HUVEC and fractalkine-transfected CHO cells at arterial (1000/s) and venous (100/s) shear rates.

Supplemental References

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Walter U, Bauersachs J. Novel role of the membrane-bound chemokine fractalkine in

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K, Schiemann M, Kennerknecht E, Sauer S, Schulz C, Kerstan S, Rudelius M, Seidl S,

Sorge F, Langer H, Peluso M, Goyal P, Vestweber D, Emambokus NR, Busch DH,

Frampton J, Gawaz M. Platelets secrete stromal cell-derived factor 1 and recruit

bone marrow-derived progenitor cells to arterial thrombi in vivo. J Exp Med.

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Legends to Supplemental figures

Supplemental Figure 1: Activated endothelial cells surface express fractalkine. a) Primarily isolated and cultivated HUVEC (resting or stimulated with 50 ng/mL TNF- and

20 ng/mL IFN- for 20hrs) were assessed for their surface expression of membrane-bound fractalkine (CX3CL1) by flow cytometry (n=3; **P<0.001 vs. resting). b) HUVEC (resting or stimulated with 50 ng/mL TNF-, 20 ng/mL IFN- or both for 20hrs) were assessed for

CX3CL1-surface expression by flow cytometry (n=3; *P<0.05, **P<0.001 vs. resting). c)

Representative FACS histogram showing fractalkine (CX3CL1) expression by resting and

TNF- or IFN- -stimulated HUVEC. d) Representative anti-CX3CL1 immunoblotting experiments of fractalkine protein expression. The assessment of actin served as a loading control.

Supplemental Figure 2: Surface expression of adhesion molecules on primarily isolated and cultivated on HUVEC.

Primarily isolated and cultivated HUVEC (resting or stimulated with 50 ng/mL TNF- and 20 ng/mL IFN- for 20hrs) were analyzed for surface expression of ICAM-1, P-Selectin, E-

Selectin and control IgG by flow cytometry (n=3; *P<0.05, ** P<0.001 vs. resting).

Supplemental Figure 3: Fractalkine-induced platelet degranulation triggers platelet- leukocyte aggregate formation. a) Human whole blood was stimulated with recombinant fractalkine (1 µg/mL) for 15 minutes in the absence and presence of a selectively antagonizing fractalkine antibody (anti-CX3CL1).

Thereafter, platelet leukocyte aggregate formation was determined by flow cytometric analysis of platelet (CD41) positive cells within the leukocyte population, the latter defined by

CD11b-positivity. Aggregate formation (CD11b+CD41+ events) is given as percentage of resting, non-stimulated platelets (n=6; *P<0.05). b) Representative FACS dot blots showing platelet (CD41+)-leukocyte (CD11b+)-aggregate formation in resting and fractalkine- stimulated human whole blood. c) Human whole blood was stimulated with recombinant fractalkine (1 µg/mL) for 15 minutes in the absence and presence of an antagonizing P- selectin antibody (anti-CD62P). Thereafter, platelet leukocyte aggregate formation was determined by flow cytometric analysis of platelet (CD41) positive cells within the leukocyte population, the latter defined by CD11b-positivity. Aggregate formation (CD11b+CD41+ events) is given as percentage of resting, non-stimulated platelets (n=6; *P<0.05).

Supplemental Figure 4: Proposed mechanism for fractalkine in the modulation of leukocyte recruitment to inflamed endothelium at arterial shear stress.

During atherogenesis fractalkine (CX3CL1) is surface-expressed on inflamed endothelium.

Platelets, which adhere to inflammatory endothelial cells via established adhesion receptors

(mainly GPIIb-IIIa and GPIbα), become activated by membrane-bound fractalkine.

Fractalkine-induced platelet activation results in platelet secretion and enhanced P-selectin surface expression. P-selectin expressed on activated platelets in turn triggers the formation of platelet-leukocyte aggregates and the recruitment of leukocytes to the endothelial surface.

Once leukocytes have tethered via platelet-expressed P-selectin they can become firmly

adherent by direct binding of fractalkine to leukocyte CX3CR1 and possibly by involvement of other adhesion molecules, i.e. cellular adhesion molecules (VCAM-1, ICAM-1).