The PU used was Tecothane TT1074A (Thermedics, Waltham, MA), a polyether polyurethane. Blood tubing modified with Poly(2-methoxyethylacrylate) (PMEA), and marketed as Terumo-X™ coated tubing, and unmodified polyvinyl chloride (PVC) tubing was acquired from Terumo Cardiovascular Systems (Ann Arbor MI). A mouse monoclonal antibody directed against human CD47 (B6H12) or was purchased from BD Pharmingen (Franklin Lakes, NJ). Rabbit polyclonal antibody directed against phospho tyrosine (PY350) and a mouse monoclonal antibody directed against human SIRPα (SE7C2) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 4′-6-diamidino-2-phenylindole (DAPI) was purchased from Vector Laboratories (Burlingame, CA). Tween 20 and sodium dodecylsulfate-polyacrylamide electrophoresis gels were purchased from Bio-Rad (Hercules, CA). Protease Inhibitory Cocktail Tablets were acquired from Roche (Indianapolis, IN). Immunoaffintiy agarose beads were purchased from Calbiochem (San Diego, CA). Sephadex and the enhanced chemiluminescence detection system is a product of Amersham, GE Healthcare (Piscataway, NJ). N-Succinimidyl 3-[2-pyridyldithio]-propionate (SPDP), avidin, 2-mercaptoethanol (2-ME), 12-myristate 13-acetate (PMA), and all other chemicals and solvents, unless otherwise specified, were purchased from Sigma (St. Louis, MO).
2.2. Biotinylated CD47
Plasmids encoding the extracellular domain of hCD47 or bCD47 (GenBank Accession Number NM_174708) were PCR amplified and ligated in frame with the vector pEF-BOS-XB [13
] which formed the in frame fusion of CD4d3+4 biotin at the C-terminus of the extracellular domain of CD47. This construct was transfected into CHO (−K1) cells and the secreted CD47− CD4d3 + 4 was concentrated, biotinylated at the C-terminus, and dialyzed. The protein was affinity purified using monomeric avidin and dialyzed against PBS [7
2.3. Cell Culture
All cell lines (human MDM THP-1, human promyelocytic HL-60, rat alveolar macrophage cell line NR8383) were acquired from American Type Culture Collection (Manassas, VA) and maintained as recommended. The human MDM cell line (THP-1) were grown in RPMI media supplemented with 5% fetal bovine serum and 0.05 μM 2-ME. The human promyelocytic cell line (HL-60) were grown in Modified Dulbecco’s Medium supplemented with 20% fetal bovine serum. The rat alveolar macrophage cell line, NR8383, were grown in Ham’s F12K medium supplemented with 15% fetal bovine serum. Where indicated, cells were transduced with the addition of 1.6 × 10−7 M of PMA to the media.
2.4. Casting PVC and PU Films
PU or PVC was dissolved in dimethylacetamide and tetrahyrdofuran respectively, and then solvent cast as films with thickness ranging between 159 and 220 μm as used in prior studies [14
]. Films, both PU and PVC, were then cut into appropriate sizes for in vitro
and in vivo
2.5. Appending CD47 to the PU and PVC Surfaces
We surface modified PU and PVC films (1cm2
), and the luminal surfaces of PVC tubing (1/4 × 1/16 inch), by first soaking them in a solution of 0.1% hexadecylpyridinium chloride for 1.5 hours at room temperature. Films or tubing were rinsed in water and then soaked in a solution of 2-pyridyldithio,benzophenone (PDT-BzPh) [16
](1 mg/ml) + KHCO3
(0.67 mg/ml) in water, that was acidified with 15% KH2
(150 μl/3 ml of PDT-BzPH). The films and tubing were incubated in the acidified PDT-BzPh for 40 minutes and then rinsed with diluted (1:1000) acetic acid and both sides of the film were exposed to UV irradiation from a BioRAD UV Transilluminator 2000 (analytical mode), for 15 minutes per side. The entire length of tubing was rotated over a BioRAD UV Transilluminator 2000 (analytical mode), for thirty minutes. The modified surfaces were then soaked in KHCO3
(20mg/ml) for 20 minutes and washed 5 times with reverse osmosis (RO) treated water.
Avidin (10mg/ml) was reacted with 0.58 mg of SPDP (dissolved in dimethylformamide) for 1 hour at room temperature. The SPDP treated avidin was passed through a Sephadex column. 2-pyridyldithio (PDT) groups, on the surfaces, were reduced to Thiol-groups by incubating for 5 minutes with a solution of 20 mg/ml of TCEP (1 ml/film) in degassed PBS. Films and tubing were then rinsed 5 times in degassed PBS and then reacted, overnight, with avidin that was prior treated with SPDP and filtered. The avidin-immobilized surfaces were then washed 5 times in dH2O and biotinylated CD47 (see below for details) was added to each film.
2.6. Western Blot Analysis
Cultured HL-60 and THP-1 cells, cultured in the presence or absence of anti-SIRPα IgG antibodies as detailed below, were washed three times with PBS and scraped in ice-cold lysis buffer (100 mM pH 8.1 Tris-HCl, 10 mM EDTA, 1% Triton X-114, a proportioned amount of complete protease inhibitor cocktail, 0.2 mM orthovanadate). The cellular lysates were passed through a 21-gauge needle, and the proteins were quantified using a micro BCA protein assay kit. Prior to immunoprecipitation, the cellular lysates were spun down at 10,000 g for 10 minutes. The collected supernatant was first incubated with 5 μg of anti-SIRPα antibody (SE7C2) for an hour and then, incubated with the corresponding amounts of immunoaffinity agarose beads on a rotator rack at 4°C overnight. The immunoprecipitated SIRPα complex proteins were resolved on a 4-15% gradient sodium dodecylsulfate-polyacrylamide electrophoresis gel using the method described by Laemmli [17
], and the proteins were transferred to a 0.2 μm pore size polyvinylidene fluoride (PVDF) membrane (Invitrogen, Carlsbad, CA), followed by immunoblotting for the presence of phosphorylated tyrosine using anti-phospho tyrosine antibody (PY350) at manufacturers’ recommended dilutions in 10 mM pH 7.5 Tris-HCl, 100 mM NaCl, and 0.1 % Tween 20 (TTBS) with 5% non-fat milk. The immune complexes were detected with the species-appropriate, horseradish peroxidase-conjugated secondary antibodies in recommended dilutions in TTBS with 5% non-fat milk and were visualized with an enhanced chemiluminescence detection system on X-ray films.
Where indicated, the autoradiographs were scanned, imported to the Quantity One version 4.6.9 (Biorad, Hercules, CA) program and individual bands were automatically separated using the band attributes function. To determine the relative intensity, the histogram function in Adobe Photoshop (Adobe, San Jose, CA) was used to measure the mean level of grey scale intensity. This value was subtracted from the background value and correlated to the loading control band of the same sample.
2.7. Cell Attachment Studies (in vitro)
THP-1, HL-60, or NR8383 cells were differentiated with the addition of PMA to their respective media and were cultured on PU or PVC films for a predetermined period of time. Additional studies examined the binding of activated HL-60 to PVC tubing or PVC tubing that was modified by surface immobilized CD47 (via avidin-biotin affinity) or avidin modified PVC tubing (avidin control). The tubes were capped at both ends and shaken for 3 hours at 37°C. At the conclusion of the protocol, films or tubing were washed and remaining cells were fixed with 4% paraformaldehyde and stained with the nuclei specific stain DAPI. Cell retention was quantified by staining with the fluorescent dye DAPI (blue color on fluorescent micrographs) and visualized using a fluorescent microscope with the appropriate filter set and separate 200X fields were counted. Where indicated cells were cultured in the presence of anti-SIRPα antibody (SE7C2) directed against the protein’s extracellular domain, or IgG control.
2.8. Rat Subdermal Implants
Male Sprague Dawley rats, weighing between 300 and 350 grams were used for subdermal implant studies as previously described [18
]. Rats were anesthetized with isoflurane and administered Flunixamine post surgery for analgesia. Each of the five rats per group received three 1 cm2
PU-films that were composed of unmodified PU or PU surface modified with covalently appended human CD47 (hCD47) or bovine CD47 (bCD47). At the 70-day termination of the study, rats were euthanized by carbon dioxide asphyxiation and the subdermal implants were removed, rinsed briefly with saline and further processed as detailed below. All procedures and animal husbandry were in compliance with NIH standards pertaining to the care and use of laboratory animals as approved by the IACUC of the Children’s Hospital of Philadelphia.
2.9. Assessment of hCD47 from explanted PU films
Following explantation, PU films were placed into 0.5 ml of a 1 % SDS solution and sonicated. The films were then transferred to a fresh solution of 1% SDS and sonicated. The films were then transferred to a 0.5 ml of PBS and sonicated. All sonicates were pooled from each film and vacuum dried. The resulting pellet was resuspended in PBS and processed for Western blotting analysis as mentioned above using the human CD47 specific antibody (B6H12).
2.10. Environmental Scanning Electron Microscopy (ESEM)
Images were acquired from FEI XL30 field emission ESEM (FEI, Hillsboro, OR). Samples were cooled to 5°C using a Peltier cooling stage and pressure was maintained between 4-5 torr to achieve a relative humidity of approximately 70%.
2.11. Fourier transform infrared spectroscopy-attenuated total reflectance
Explanted PU films were processed for FTIR as previously described [18
]. Briefly, explanted films were washed in a 1% Triton X-100 in PBS solution followed by a second series of washes in PBS. A final rinse in 70% ethanol was performed to remove residual detergent. The films were then scanned with Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) spectroscopy using a Nicolet 5-Protègé 460 spectrophotometer E.S.P. (Nicolet Madison, WI). All spectra were obtained under identical conditions from 200 scans collected at a resolution of 2 cm−1
, at a 45° angle of incidence and the atmospheric water vapor and carbon dioxide were accounted for by subtracting the appropriate reference spectrum using the Omnic software package. Deconvoluted peak measurements were acquired for the 1174 cm−1
peak and normalized to the measured 1590 cm−1
peak heights [14
2.12. Statistical Analysis
Data were calculated as means ± standard error (SE). Student’s t-test was used to determine the significance of differences. Statistical significance was noted with p≤0.05