Agonist-regulated trafficking of NADPH oxidase
SUPPLEMENTARY DATA
Patrick Ejlerskov, Dan Ploug Christensen, David Beyaie, James B. Burritt, Marie-Helene Paclet, Agnes Gorlach, Bo van Deurs, and Frederik Vilhardt.
NADPH oxidase Is Internalized by Clathrin-Coated Pits and Localizes to a Rab27A/B-Regulated Secretory Compartment in Activated Macrophages
Table of contents:
Supplementary Materials and Methods
Supplementary Figure 1
Supplementary Figure 2
Supplementary Figure 3
Supplementary Figure 4
Supplementary Figure 5
Supplementary MOVIE 1
Supplementary Materials and Methods
Reagents
Antibodies used (continued); rat anti-grp94 mAb (Stressgen; Farmingdale, USA), rat anti-mouse CD11b mAb (Serotec; Oxford, UK), rat anti-mouse CD16/CD32 mAb (BD Biosciences; Franklin Lakes, NJ, USA), monoclonal anti-clathrin IgM (Research Diagnostics; Danvers, MA 01923, USA), polyclonal rabbit anti-clathrin antibodies (Abcam; Cambridge, UK), anti-transferrin receptor mAb (Zymed; San Francisco, CA 94080, USA), anti-transferrin receptor mAb recognizing external epitope (Sanquin; Amsterdam1006 AD, Netherlands), polyclonal rabbit anti-Rab5 (Abcam and Epitomics (Burlingame, CA 94010-1303, USA.)), polyclonal rabbit anti-Rab11 (Abcam and Cell Signaling), monoclonal anti-EEA1 mAb (Santa Cruz; Santa Cruz, CA.,USA), anti-GFP mAbs (Boehringer-Mannheim (Basel, Switzerland)and Molecular Probes (Grand Island, NY, USA))and anti-sortilin antibodies (Abcam).
About antibody specificity:
For most of the morphological and biochemical work two antibodies directed against cyt b558 has been used; polyclonal rabbit anti-p22phox (FL195, Santa Cruz) and anti-gp91phox mAb 54.1(1). FL195 in our hands is a very specific and reliable antibody for both immunofluorescense and western blotting applications. The mAb 54.1 can be used for detection of NOX1, NOX2 (gp91phox), and likely NOX3 and NOX4 by western blotting(2)and has been used for immunolocalization of NOX2 (3) in phagocytes and other cell types. In phagocytes gp91phox is the predominant form. We find mAb 54.1 is highly specific in western blotting applications (of phagocytes), but in morphological assays sometimes gives dubious immunoreactivity depending on cell type, perhaps due to cross-reactivity with an ER-residing disulfide isomerase protein GRP58(2). However, we did not observe ER staining with mAb 54.1 in the phagocyte cells used for this study. The mAb 54.1 does not recognize non-denatured antigen in intact or saponin-permeabilized neutrophils, and only partially affects immunosedimentation of purified and detergent-solubilized cyt b558(1). In accordance with these observations we find that paraformaldehyde fixation (together with saponin permeabilization) is required for specific immunoreactivity in immunofluorescence applications. Additionally, we suspect, as originally suggested(1), that the 54.1 mAb epitope on gp91phox is partially shielded in vivo due to interactions with other proteins.
BMDM purification and culture
To obtain bone marrow-derived macrophages (BMDMs) marrow from the femurs of several rats (200-250 gram) was flushed out with a 21G-needle with PBS containing 2% fetal calf serum (FCS, Gibco) and 1% penicillin/streptomycin (P/S). Subsequent to centrifugation at 800g for 5minutes, cells were resuspended in erythrocyte-lysis buffer, 50 mM NHAc pH 7.4, for 1 minute, before addition of a tenfold excess of PBS. The procedure was repeated to eliminate all erythrocytes (usually two-three times). BMDMs were then seeded in 6-wells (Nunclon) at a density of 3×106 cells/well in RPMI-1640 with 10% FCS, 1%pyruvate, 2%Non essential amino acids, 10 M-mercaptoethanol, and 1% penicillin-streptomycin.Cells were incubated with 10-20 ng/mlmacrophage colony-stimulating factor (M-CSF) for 8-10 days with intermittent changes of half of the medium and the full complement of M-CSF. At the end of the differentiation period, macrophages were subsequently gently pipetted of the wells in PBS, counted, and reseeded in 8 well glass chamber slides at a density of 100.000 cells/well and used next day for experiments.
Peritoneal macrophages purification and culture
Resident peritoneal macrophages were collected from rats 6 months or more of age by peritoneal lavage with 10 ml of phosphate-buffered saline containing 5% glucose injected and collected with a 21G needle. Usually 6-8 ml could be obtained. After centrifugation any red blood cells visible in the cell pellet were removed by hypotonic lysis as above. Remaining cells were distributed into 10 cm petri dishes in RPMI-1640 growth medium for one hour to allow adherence of macrophages followed by 1-2 gentle rinses in growth medium to remove non-adherent cells. Next day macrophages were redistributed to 6-wells or 8-chambers for experiments by gentle pipetting inPBS buffer.
Purification of human monocytes
Buffy coats obtained at the university hospitalwere diluted 1:1 with PBS containing 3% FCS, and then fractionated by standard Hypaque-Ficoll centrifugation. The mononuclear cells were washed twice in PBS, and then monocytes were enriched by differential adhesion in RPMI1640 medium for one hour at 37ºC. Alternatively to obtain a higher degree of purity the mononuclear band from the Hypaque-Ficoll gradient was further separated on a preformed percoll gradient (58% percoll, 20.000g, 50 minutes) by centrifugation at 400g for 25 minutes. The upper white band with monocytes was collected. In both instances, monocytes were cultured for 5-7 days without stimulation for maturation into macrophages at which time they were used for experimentation.
Supplementary Figure 1
Purity of isolated microglia, peritoneal macrophages and bone marrow-derived macrophages from rat determined by flow cytometry.
Macrophage populations isolated as described were surface-labeled with primary anti-CD11b, anti-CD32, or anti-CD11c mAbs or with isotype matched control IgG followed by Alexa488-conjugated secondary goat-anti mouse antibodies. Cells were then analyzed by flow cytometry and viable cells gated by FSC/SSC scattering. The number indicated in each panel refers to the percentage of labeled cells of the total cell population.
Supplementary Figure 2
Peritoneal and bone marrow-derived macrophages from rat redistribute cyt b558 from cell surface to intracellular vesicles upon LPS stimulation.
Resident peritoneal macrophages or bone marrow-derived macrophages purified as described were stimulated with 1 g/ml or 100 ng/ml LPS, respectively, overnight before paraformaldehyde fixation and permeabilization forimmunofluorescense labeling using rabbit polyclonal anti-p22phox antibodies (green) and a mouse anti-CD11b mAb (red) followed by appropriate secondary fluorophore-conjugated antibodies. Note that p22phox, but not CD11b, is redistributed to an intracellular position in both peritoneal macrophages (pMø) and bone marrow-derived macrophages (BMDM) by LPS stimulation. Bars, 10 m.
Supplementary Figure 3
Deprival of GM-CSF causes slow redistribution of cyt b558 from intracellular stores to the cell surface in Ra2 microglia
A) Ra2 microglia were cultured in the absence of GM-CSF for the indicated time and then surface-labeled with biotin. After cell lysis and streptavidin precipitation of surface-resident proteins equal aliquots were western blotted with anti-gp91phox antibodies. B) Mean and SEM of optical density of gp91phox bands were derived from three independent experiments. C and D) Ra2 microglia were cultured in the presence (control; C) or absence of GM-CSF (D) for twelve days before fixation and processing for immunofluorescence using anti-gp91phox mAb 54.1 (green) and rat anti-CD16/32 (red). Note the increased cell surface expression and co-localization of gp91phox with CD16/32 in GM-CSF-deprived cells (arrows). E and F) Ra2 microglia over-expressing human gp91phox were cultured in the presence (control; E) or absence of GM-CSF (F) overnight before fixation and immunofluorescence as above. Note again increased surface expression and co-localization with CD16/32 in the absence of GM-CSF (arrows).
Supplementary Figure 4
Cyt b558 is redistributed to phagosomes regardless of cell surface or intracellular localization
Primary microglia purified as described were left alone (control) or treated with 100 ng/ml LPS overnight. Subsequently, they were challenged with TXR-conjugated zymosan particles for 15 minutes before fixation and processing for immunofluorescence as indicated with either anti-gp91phox mAb 54.1 or rat anti-CD11b mAb. Top panel shows cells before zymosan challenge, the lower panel after zymosan phagocytosis. Note that phagosomes receive cyt b558 in either case.
Supplementary Figure 5
Mutation of potential consensus signals for clathrin-mediated endocytosis in gp91phox does not influence its subcellular distribution in COS cells.
Sorting signals 456DLLQLLand 201YFEV were mutated in CFP-gp91phox (underlined amino acid position changed to alanine) by site directed mutagenesis. Shown is COS cells expressing p22phox and transduced with pLenti-CFPgp91phox vector coding for wild type, LL, or YFEV mutant. Note that the distribution of wild type and the LL mutant distribute similarly (plasma membrane and recycling compartment, the latter marked by arrows) while the YFEV mutant seemed impaired in rER exit. Bars, 10 m.
Supplementary Video 1
Time-lapse fluorescense recording of CFP-gp91phox exocytosis after ATP stimulation of Ra2 microglia.Using a Zeiss LSM510 confocal laser microscope with a heated stage,medial, confocal sections (1.0 m) of CFP-fluorescence in Ra2 microglia expressing CFP-gp91phox were acquired every 18 seconds with a 63X, NA 1.2 oil objective, before and after ATP stimulation (1 mM), for a total of 32 minutes. Time point of ATP-addition (7 minutes)is indicated in the movie.The large, round structures void of CFP-gp91phox fluorescence (besides the nucleus) are macropinosomes.Shown are two movies at different magnification to show population phenotype and detail, respectively.
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