www.sciencemag.org/cgi/content/full/1120225/DC1

Supporting Online Material for

A Role for the Phagosome in Cytokine Secretion Rachael Z. Murray, Jason G. Kay, Daniele G. Sangermani, Jennifer L. Stow*

*To whom correspondence should be addressed. E-mail: j.stow@imb.uq.edu.au

Published 10 November 2005 on Science Express DOI: 10.1126/science.1120225

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S3 Table S1 References

Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/1120225/DC1)

Movies S1 to S7

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Supporting Online Material Materials and Methods Antibodies and reagents. Rabbit polyclonal and rat monoclonal anti-mouse TNFα antibodies were purchased from Calbiochem and Auspep respectively. Mouse monoclonal PE-conjugated anti-TNFα antibody was purchased from BD Biosciences. Rabbit polyclonal antibodies specific for VAMP3 were purchased from AbCam, while rabbit polyclonal antibodies specific for Rab11 and mouse monoclonal antibodies specific for transferrin receptor were purchased from Zymed. Rat monoclonal antibodies specific for LAMP1 were purchased from Southern Biotech, while rabbit polyclonal antibodies specific for VAMP8 were purchased from Synaptic Systems and rabbit polyclonal anti-TACE antibodies were purchased from Chemicon. Mouse monoclonal antibodies specific for syntaxin 6 and Vti1b were purchased from Transduction Laboratories. Rabbit polyclonal antibodies specific for syntaxin 4 were a kind gift from David James (Garvan Institute of Medical Research, Sydney, Australia). Mouse monoclonal antibodies specific for tubulin, Alexa-488 conjugated phalloidin (used to label F-actin), Texas Red and Alexa 647-conjugated transferrin (Tfn) and DilC18(5)-DS lipophilic dye were purchased from Molecular Probes. HRP-conjugated Tfn was purchased from Rockland. 3,3’-diaminobenzidine (DAB) was purchased from Sigma-Aldrich. TACE inhibitor, TAPI-1, was purchased from Calbiochem. LPS from Salmonella minosota Re595 was purchased from Sigma. IFNγ was purchased from R&D Systems. GFP-tagged constitutively active Rab11a (Rab11Q70L-GFP) and dominant negative-Rab11a (Rab11S25N-GFP) were provided by Rob Parton (University of Queensland, Australia) with permission from Marino Zerial (Max Planck Institute for Molecular Cell Biology and Genetics, Germany). GFP-tK was a kind gift from John Hancock (University of Queensland, Australia) and ApoE-GFP was a kind gift from Len Kritharides and Wendy Jessup (Centre for Vascular Research, NSW, Australia). Candida albicans was kindly provided by Robert Ashman (University of Queensland, Australia). Cell culture, phagocytosis, molecular cloning and electroporation. RAW264.7 mouse macrophages were cultured, electroporated and activated with LPS (1-2 hours) as previously described (1). In some experiments macrophages were primed for 18 hours in the presence of 500 pg/ml IFNγ prior to their incubation with LPS (100 ng/ml) for 2 h or incubated with live Candida albicans at a ratio of 10:1 (yeast:macrophage) for 15-40 min. Where stated 10 µM TACE inhibitor was added to the cells at the same time as the LPS or Candida albicans. VAMP3 (full-length GFP-VAMP3 and truncated GFP-VAMP3, aa 1-81) was subcloned from a NIA clone (NIA ID H3025H09) into the pEGF-C2 vector (Clontech, BD Biosciences) to produce an N-terminal GFP-tagged protein. TNFα was subcloned into pEGF-C2 vector (Clontech, BD Biosciences) from an existing TNFα clone (2) to produce an N-terminal GFP-tagged protein. Macrophages were electroporated, using the Gene Pulser II (Bio-Rad), for transient expression of cDNAs using 2.5 × 107 cells with 10 µg DNA, with a high capacitance setting (280 mV and 950 µF). Cells were washed and typically cultured for 4-24 hrs.

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Small interfering RNA (siRNA) treatment. RAW264.7 cells, plated on glass coverslips, were transfected with three different siRNA constructs designed to silence mouse VAMP3, (Silencer Validated siRNA ID#186988-90, Ambion) using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen) and cultured for 24 hours, retransfected under then same conditions then cultured for a further 24 hours prior to LPS treatment in the presence of TACE inhibitor. Microarray data analysis. RNA was harvested from RAW 264.7 macrophages, cultured in the presence of LPS (100 ng/ml) for 0, 2 or 12 hour. The RNA was probed using the Affymetrix 430A mouse gene chip as previously described (1); results published at GEO, accession number GSE1459, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1459). SDS-PAGE, immunoblotting, immunoprecipitation and immunofluorescence staining. Raw264.7 cells were washed three times with PBS, scraped and lysed in Buffer A (10 mM Tris, pH 7.4, containing 1 mM EDTA, 0.5% Triton X-100, and Complete protease inhibitors (Roche Applied Science)), by passage through a series of successively smaller needles and centrifuged for 10 min at 17,000 X g. The supernatant was assayed for protein content (BioRad protein assay), subject to SDS-PAGE separation and analyzed by immunoblotting (1). For immunoprecipitation RAW264.7 cell lysates were incubated with antibody (5 µg) bound to protein A-Trisacryl (Pierce) for 2 h at 4 C. Beads were then washed with excess Buffer A containing 150 mM NaCl and bound proteins were solubilized in SDS-PAGE sample buffer. Immunofluorescence staining was performed as described previously (1). Assays for TNFα trafficking, surface delivery and secretion. The trafficking of TNFα from the Golgi complex to the cell surface was measured using an immunofluorescence-based assay as previously described (3). Briefly, macrophages were incubated in the presence of LPS (100 ng/ml) and TACE inhibitor (10 µM) for 2 hours, fixed with 4% paraformaldehyde and immunostained to label surface TNFα, then permeabilized with 0.1 % Trition X-100 and immunostained for internal TNFα. A commercial enzyme-linked immunosorbant assay (ELISA) kit (BD OptEIA, BD Biosciences) was used according to the manufacturer’s instructions to determine levels of secreted TNFα. HRP inactivation assay. An HRP inactivation assay was modified from the protocol of Ang et al (4). RAW264.7 cells were incubated with Tfn-HRP (10 µg/ml) and Tfn-Alexa-647 (10 µg/ml) in media for 1 hr in the dark at 37°C in the presence of 100 ng/ml LPS. Cells were washed twice in ice-cold PBS and surface-bound Tfn-HRP was removed by two 5 min washes with 0.15 M NaCl and 20 mM citric acid, pH 5. Cells were then washed twice with ice-cold PBS and incubated in the dark for 1 hr with PBS containing 0.1 mg/ml DAB and 0.025 % H2O2 to the inactivation sample (the control contained PBS alone). Cells were washed twice in PBS containing 1 % BSA to stop the reaction and then incubated in pre-warmed media containing LPS and TACE inhibitor for 2 hrs at 37°C. Cells were washed, fixed and immunostained for either surface or internal TNFα.

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Cell surface labelling and FACS analysis. Raw264.7 macrophages were electroporated with either GFP-alone or GFP-VAMP3, cultured for 24 hours, stimulated with LPS for 2 hours and the cell surface was labeled, according to the manufacturer’s instructions, with 2 µM DilC18(5)-DS fluorescently labeled lipophilic dye (Molecular Probes) for 2 min followed by a 15 min incubation on ice. Cells were washed three times with ice-cold PBS and fixed using 4% paraformaldehyde. Confocal microscopy confirmed that the lipophilic dye labeling was restricted to the cell surface. Immunofluorescence analysis was performed using the FACSVantage SE DiVa (Becton Dickinson) and the subsequent data was analyzed using WinMDI software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA). The relative amount of DilC18(5)-DS lipophilic dye on the plasma membrane was used as an indicator of cell surface area and was assessed on the basis of mean fluorescence intensity (MFI), in arbitrary units. Transfection itself did not alter cell size. Live cell imaging. RAW264.7 cells cultured on glass bottom microwell dishes (MatTech, USA) were imaged in CO-2 independent media (Gibco, Invitrogen) at 37°C using a heated stage on a Zeiss LSM510 META confocal microscope (Carl Zeiss Microscope Systems, Germany) equipped with a 60 or 100 X oil objective. Movie frame capture rates were between 1.5 and 12 seconds, with total capture periods ranging from 5 to 40 minutes. Movies were cropped, constructed and analyzed using Zeiss LSM software version 3.2, and exported as Quicktime movies with playback speeds of 8-10 frames per second. Scanning Electron Microscopy. RAW264.7 cells cultured on 12mm glass coverslips were fixed in 2% glutaraldehyde in cacodylate buffer, post-fixed in 1% osmium tetroxide (ProSciTech, Queensland, Australia) and dehydrated through a graded series of ethanols (5). The coverslips were washed twice in hexamethyldisilazane (HMDS, ProSciTech), air dried overnight and then platinum coated using a Baltec Med020 coater (Bal-Tec, Liechtenstein) before viewing on a JEOL 6300 Field Emission Scanning Electron Microscope (Jeol Australasia, Brookswater, NSW, Australia) at 7kV. Images were captured using ImageSlave software (ImageSlave, Sydney, Australia).

GFP-VAMP3 Rab11

Figure S1. VAMP3 localizes to the RE. Macrophages expressing GFP-VAMP3 werestimulated with LPS for 2 hours, fixed, permeablized and immunostained for the REprotein Rab11.

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Figure S2. Increased cell surface in activated macrophages. (A) LPS-treated anduntreated macrophages expressing either GFP or GFP-VAMP3 were briefly incubatedwith the lipophilic dye DilC to label the cell surface. Cells were then fixed and analysedby FACS. Increasing the level of VAMP3 increased cell surface area. (B) LPS-activatedand control macrophages were fixed and analysed by scanning electron microscopy.Control cells have sparse surface features while activated cells have abundant surfaceruffles which increase the surface area. (C) GFP-VAMP3 staining on the cell surfaceis seen concentrated on ruffles. Bars = 5 μM.

GFP-VAMP3

5 μm

5 μm

Figure S3. Model showing delivery of TNFα from the RE to the phagocytic cup and itssubsequent release from the cell surface. The newly-synthesized, transmembrane precursor ofTNFα is delivered to the RE which contains the R-SNARE VAMP3. The RE membrane fuses withthe plasma membrane at the site of a nascent phagocytic cup where the cognate Q-SNARE complex(syntaxin 4-SNAP23) for VAMP3 is concentrated. This expands the membrane to accommodatephagocytosis while simultaneously delivering transmembrane TNFα to the cell surface. TACE, theenzyme that cleaves TNFα, is strategically positioned at the cup to ensure rapid cleavage and releaseof soluble TNFα. Soluble TNFα is released from the membrane before closure of the cup and ittherefore does not appear with the ingested microbe in the mature phagosome. This trafficking stepensures delivery of both membrane and cytokine to the surface for early inflammatory responses.

VAMP3TNFα

Syntaxin 4TACE

RE

TransmembraneTNFα is deliveredto the phagocytic

cup via the RE

Soluble TNFα isreleased from the cell

surface and isexcluded from thecup before closure

TNFα delivery to and release from the phagocytic cup

VAMP3

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Movie S1. Newly synthesized TNFα is trafficked to the RE. GFP-TNFα was expressed and imaged in live LPS-stimulated macrophages at a focal plane through the Golgi complex on a Zeiss LSM510 META confocal microscope. Newly synthesized GFP-TNFα concentrates in the Golgi region and GFP-TNFα-containing carriers move centripetally towards and fuse with a larger compartment (1-2 µm diam) that resembles in size and shape the VAMP3-positive REs labelled elsewhere in this study (boxed area) (captured at 1.52 second intervals over 34 seconds). Movies S2 and S3. TNFα is trafficked through a transferrin-positive RE. GFP- TNFα (green) was imaged in live LPS-stimulated macrophages after uptake of Alexa-647-conjugated Tfn (red) as a marker of the RE. GFP-TNFα enters a Tfn-positive RE (boxed area), remains there transiently and then another GFP-TNFα carrier exits the RE and moves along a trajectory towards the plasma membrane (captured at 2.2 second intervals over 30 seconds). Movie S4 and S5. Newly synthesized TNFα is delivered to the phagocytic cup. GFP- TNFα was imaged live in Candida albicans-phagocytosing, activated macrophages, movie frames captured at 8.5 second intervals over 300 seconds (movie 4) and 1700 seconds (movie 5). Movie 4 shows a sequence with GFP-TNFα in intracellular compartments and carriers and concentrated in the phagocytic cup as it forms around a yeast particle. GFP-TNFα on the membrane of the phagocytic cup surrounds the yeast and is then left concentrated at an outer point as the yeast is ingested. Movie 5 shows GFP-TNFα moving towards and being delivered to a nascent phagocytic cup. Movie S6. Newly synthesized ApoE is not delivered to the phagocytic cup. GFP-ApoE was imaged in Candida albicans-phagocytosing macrophages (frames captured at 1.4 second intervals for 140 seconds), showing that GFP-ApoE leaving the Golgi does not move towards the phagocytic cup during phagocytosis. Two instances can be seen of carriers containing GFP-ApoE moving towards an area of the plasma membrane away from the phagocytic cup. Movie S7. GFP-VAMP3 membranes associate with the phagocytic cup. GFP-VAMP3 was imaged in Candida alibicans-phagocytosing, stimulated macrophages (frames captured at 7 second intervals for 1225 seconds). GFP-VAMP3 labeled membranes move towards and are delivered to the phagocytic cup.

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Table S1. R-SNARE gene regulation in macrophages. Changes in gene expression after 2 and 12 hours stimulation of RAW264.7 cells with LPS are shown for R-SNARE genes present on the array.

Gene Accession number 2 hours LPS 12 hours LPS VAMP1 NM_009496 ND ND VAMP2 NM_009497 NC NC VAMP3 NM_009498 I (+1.2) I (+2.8) VAMP4 NM_016796 NC D (-1.4) VAMP5 AK009266 ND ND VAMP7 BC003764 NC NC VAMP8 NM_016794 NC NC

Key: NC is no change, ND is not detected, I is increased and D is decreased, with fold increases indicated.

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References and Notes

1. R. Z. Murray, F. G. Wylie, T. Khromykh, D. A. Hume, J. L. Stow, J. Biol. Chem. 280, 10478 (2005).

2. W. Shurety, A. Merino-Trigo, D. Brown, D. A. Hume, J. L. Stow, J. Interferon Cytokine Res. 20, 427 (2000).

3. J. K. Pagan et al., Curr. Biol. 13, 156 (2003). 4. A. L. Ang et al., J. Cell Biol. 167, 531 (2004). 5. S. Seveau, H. Bierne, S. Giroux, M. C. Prevost, P. Cossart, J. Cell Biol. 166, 743

(2004).