Ethics statement
All patients donating tissue gave written informed consent. Use of tissue from lung resection was approved by the National Research Ethics Service (reference 07/MRE08/42) and bronchoscopic studies were approved by the Leicestershire Research Ethics Committee (reference number 4977).
Human mast cell purification and culture
HLMCs were isolated and cultured as described previously [6, 10]. Final purity was >99 %, and viability >97 % in all experiments.
The leukaemic human MC line HMC-1 (Dr J Butterfield, Mayo Clinic, Rochester, USA) was cultured as described previously [6].
Human airway smooth muscle cell and BEAS-2B culture
HASMCs were isolated using explant culture of ASM bundles as previously described [11]. HASMCs were cultured in DMEM supplemented with 10 % FBS, antibiotic/antimycotic agents and non-essential amino acids [11]. BEAS-2B epithelial cells were purchased from the European Collection of Animal Cell Cultures (Porton Down, Wiltshire, UK). Cells (passages 8–12) were grown on human plasma fibronectin-coated T75 culture flasks in BEBM media (Clonetics Cat. No. CC4175), with an added enhancement bullet kit (Clonetics Cat. No. CC4175), Pen/Strep (5 ml) and fungizone (5 ml) to create basal epithelial growth media (BEGM). BEAS-2B were then passaged on to human plasma fibronectin-coated 96-well plates and then grown to confluence prior to use in adhesion assays as described previously [6].
Micro-organisms and plasmids
Escherichia coli XL1-blue (endA1 gyrA96 (nalR) thi-1 recA1 relA1 lac glnV44 F’[::Tn10 proAB+ lacIq Δ (lacZ) M15] hsdR17 (rK
− mK
+)) and HB2151 (K12, ara, del (lac-pro), thi/F’proA + B+, lacIq, lacZdelM15) were obtained from Pharmacia LKB (St Albans, Herts, UK). Gene Hogs competent cells (F- mcrA Δ (mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ (ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2 (confers phage T1 resistance) were obtained from Invitrogen (Paisley, UK). Ex-12 helper phage [12] was obtained from Ig Therapy Co. (College of Agriculture of Life Sciences, Kangwon National University, Republic of Korea). The construction of the phagemid vector pSD3 is described by Li et al. [13].
Preparation of HLMC and HMC-1 membranes
HLMCs from several donors were suspended in buffer (10 mM Tris–HCl, pH 7.4, 1 mM EDTA, 200 mM sucrose, 1 mM phenylmethylsulfonyl fluoride) and homogenized. The nuclei and cell debris were removed from the homogenate by centrifugation (900 g, 10 min, 4 °C). The pellet was washed with buffer and centrifuged (900 g, 10 min). The resulting supernatants were pooled and centrifuged (110000 g, 75 min, 4 °C). The supernatant was discarded, the membrane pellet dissolved in PBS, and protein concentration was measured (Bio-Rad DC protein assay, BioRad, Hemel Hempstead, UK). 1.54 mg of HLMC membrane protein was obtained from 6.5 × 107 cells. Using the same protocol, 4.6 mg of membrane protein was obtained from 1 × 108 HMC-1 cells.
Immunisation of rabbits with mast cell membranes
Two female New Zealand white rabbits were immunised with a 1 ml mixture of 0.4 mg HLMC membrane protein in 0.5 ml PBS and 0.5 ml Titermax Gold. Three weeks later, both rabbits were boosted with a mixture of 0.25 mg HLMC membrane protein and 0.5 mg HMC-1 membrane protein. After a further two weeks, both rabbits were boosted with 0.7 mg HMC-1 membrane protein. After a further two weeks, both rabbits were boosted with 0.12 mg HLMC membrane protein and 0.5 mg HMC-1 membrane protein. Post-immune sera were labelled R91 and R92.
Experiments using rabbits were carried out within the Biomedical Services Unit at the University of Leicester, UK. All animal procedures were performed under Home Office (UK) and local ethical review committee approval and compliant with the Animal (Scientific Procedures) Act 1986. The rabbits were housed in scanabur rabbit cages and fed on SDS Stanrab diet. They were on a light cycle of 12 h light, 12 h dark (7 am-7 pm). Local anaesthetic cream was applied prior to blood sampling. Terminal procedures: overdose of anaesthetic with exsanguination to confirm death.
Generation of an immune rabbit scFv-phage library
Protocols for DNA manipulation were taken from Sambrook et al. [14] or were those recommended by the manufacturers. The construction of an immune phage-display single chain variable region (scFv) antibody library was carried out as described by Gough et al. [15] and Kuhne et al. [16]. Briefly, the spleens from both rabbits were cut into thin pieces, submerged in RNAlater and stored at 4 °C. Total RNA was isolated from spleen tissue using a RNA isolation kit (Qiagen, Surrey, UK) and used in the production of first strand cDNA using a cDNA synthesis kit (Pharmacia, Herts, UK) and a random hexa-nucleotide primer. The VL and VH repertoire of the rabbits were amplified by PCR. Purified VL PCR products were cleaved with SfiI and purified VH PCR products cleaved with PflMI. Cleaved products were purified by gel extraction and the VL repertoire ligated into SfiI cut pSD3 [13]. VL-pSD3 ligation products were dialysed against sterile water and then 42 ng used to transform GenHogs OneShot competent cells. Multiple transformations were carried out yielding 3 × 106 VL-pSD3 containing cells. VL fragment diversity was assessed by amplification of the VL gene using primers pSDF (5’-TATTTCAAGGAGACAGTC-3’) and pSDseq2 (5’-AACCCACTCCTTGGCCTTC-3’) followed by digestion of the PCR products with BstNI restriction enzyme. Resulting fragments were analysed on a 3 % (w/v) agarose gel. The ‘VL library’ had an estimated diversity of 2.3 × 106 distinct light chains. The VL-pSD3 library was cleaved with PflMI and the VH repertoires ligated in. Purified pSD3:scFv ligation product (40 ng) was then used to transform electrocompetent GenHogs OneShot cells. ScFv fragment diversity was assessed by amplification of the scFv gene using primers pSDF and pSDR (5’-ATTGGCCTTGATATTCAC-3’) followed by analysis of DNA fragments produced upon BstNI digestion. Multiple transformations yielded a scFv library consisting of an estimated 3.7 × 107 different antibodies. The library DNA was purified and transformed into XL1-blue host cells. The resulting clones coded for scFv-pIII fusion proteins containing poly-Histidine and C-myc tags between the scFv and pIII domains. An amber top codon was situated between the C-myc tag and the pIII gene [13].
Selection of mast cell-specific scFv-displaying phage
Phage were prepared from the rabbit anti-mast cell library as described by Oh et al. [12] utilising Ex-12 helper phage to rescue scFv-displaying phage. Phage selection was carried out in solution; HMC-1 cells were pelleted at 500 g and washed twice in RPMI (Sigma, Poole, UK), re-pelleted and then resuspended in 1 ml Hank’s buffered salt solution (HBSS, 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM, MgSO4, 4.2 mM NaHCO3). The phage library was then depleted of antibodies that display binding to antigens on the cell surface that are not recognised by the immune sera; this was carried out by binding the phage scFvs to HMC-1 cells that had been epitope-masked as described by Popkov et al. [17]. Briefly, 0.5 ml of the HMC-1 cells (1-2 × 107 cells) were incubated with 200 μl of a pool of the anti-mast cell serum R91 and R92 for 30 min at room temperature before cells were blocked with 2 % (w/v) Marvel-HBSS for 30 min at RT. Cells were then washed 3 times in HBSS and resuspended in 1 ml of blocked phage solution. For the initial round of panning, PEG-precipitated library phage (5 × 1013/ml, 1 ml) were blocked in 3 % (w/v) Marvel-HBSS for 45 min at RT. For subsequent rounds, 1 ml of the supernatant of an overnight bacterial culture of phage was used. Blocked phage were incubated with the epitope-masked HMC-1 cells for 30 min at room temperature with rotating. Cells were pelleted (500 g, 5 min) and the phage-containing supernatant removed (‘depleted phage’). ‘Unmasked’ HMC-1 cells (1-2 × 107 cells) were blocked in HBSS-3 % (w/v) Marvel for 30 min at RT. ‘Unmasked’ cells were then pelleted and resuspended in the ‘depleted phage’ and incubated for 1 h at RT with rotating. Unbound phage were removed by washing 3 times with RPMI and 3 times with HBSS; bound phage were then eluted in 25 mM triethylamine as described previously [15]. Up to four rounds of iterative scFv-phage selection were carried out with each round including a negative selection of phage against epitope-masked HMC-1 cells followed by a positive selection against unmasked HMC-1. Monoclonal phage-displayed scFvs were obtained after 3 or 4 rounds of panning by plating out the XL1-blue infected cells to single colonies. Cultures of these single colonies were then used to produce monoclonal scFv-phage particles by rescue with Ex-12 helper phage. For the production of soluble scFvs, monoclonal scFv-phage particles were used to infect HB2151 host cells followed by IPTG induced expression [15].
Phage-scFv ELISA
Both polyclonal and monoclonal phage preparations were assayed against HMC-1 cells by ELISA. Polyclonal phage were assayed at 1 × 1011/ml and monoclonal phage were assayed using 100 μl of supernatant from an overnight culture of bacteria following phage rescue. All steps were carried out within deep well tissue culture plates and the final step transferred to Nunc Maxisorb ELISA plates (Gibco BRL, Nunc products) to allow absorbance readings to be taken. All blocking and wash steps were as in the panning protocol. Phage-scFv were bound to HMC-1 cells (~106 cells per well) for 1 h at room temperature, bound phage were detected in two steps using biotin labelled anti-fd antibody (1/160) and then extravadin-AP conjugate (1/10,000; both from Sigma). Signals were developed with PNPP substrate from Sigma and the absorbance at 405 nm measured.
ScFv purification
Soluble scFv was purified via its poly-His tag on a HisPur Cobalt resin column as recommended by the manufacturers (Pierce). ScFv concentration was estimated using the BCA protein assay kit (Pierce, Rockford, IL, USA) and protein purity assessed by analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Pure scFvs were stored in 37.5 % (v/v) glycerol at −80 °C at concentrations in the range 0.1 to 0.9 mg/ml.
SDS-PAGE and Western blotting
Discontinuous SDS-PAGE was carried out through a running gel containing 12 % (w/v) total acrylamide using NuPAGE pre-cast Bis-Tris gels and Seeblue Plus 2 protein markers (Invitrogen, Paisley, UK). Electrophoresed gels were stained using 0.05 % (w/v) Coomassie Brilliant Blue R250 (Fischer Scientific). For Western blots, separated proteins were transferred to polyvinylidine difluoride membrane (Roche) using a NuPAGE Blot module (Invitrogen) at 30 V for 1 h. The 9E10 anti-C-myc antibody was used to identify scFvs. For staining of Kit in Western blots, a mouse IgG1 anti human Kit antibody (E1, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used.
Identification of scFv amino acid sequences
ScFv clones were sequenced by dye termination with AmpliTaq DNA polymerase, FS on a 377 ABI automated DNA sequencer using the primers pSDF (5’-TATTTCAAGGAGACAGTC-3’) and pSDR (5’-ATTGGCCTTGATATTCAC-3’). The scFv sequences were aligned as described by Kabat et al. [18]. The antibodies consisted of a VH-a1 heavy chain [19] combined with a kappa light chain.
Flow cytometry
MCBS1 mouse mast cells were a kind gift from Dr Dean Metcalfe, National Institute for Allergy and Infectious Diseases, NIH, Bethesda, MD) [20]. Control non transfected cells, mock transfected cells (E1-AA685) or human Kit-transfected cells (W1-AA677) were stained with 4 ug/mL PE-labelled anti-Kit mAb (BD Bioscience, Oxford, UK) or 5 μg/mL A1 scFv antibody followed by 9E10 (anti-myc) secondary antibody, which was then indirectly labelled with R-Phycoerythrin (PE)-labelled rabbit anti-mouse antibody (Dako, UK). Appropriate isotype controls were performed (mouse mAb IgG1-PE (BD Bioscience, Oxford, UK) or E4 scFv isotype). Staining was analysed by one colour flow cytometry on a FACSCanto (BD Biosciences, Oxford, U.K.). The same protocol was used for analysis of scFv binding to HMC-1 cells and HLMCs where bound scFv was detected with anti-C-myc 9E10, and then labelled with FITC-labelled rabbit anti-mouse antibody (Dako, Ely, UK), or RPE-labeled rabbit anti-mouse (Dako) as described previously [21]. HMC-1 cells were pre-incubated with SCF 100 ng/ml for 15 min to assess the effect of Kit internalisation on scFv binding.
To detect polyclonal sera binding to HLMCs, the same protocol was performed but using 105 mast cells and 10 μl of 1:10 to 1:10,000 dilutions of polyclonal sera, and using PBS-0.1 % (w/v) BSA buffer throughout. Bound polyclonal antibody was detected with anti-rabbit IgG-FITC (1:10 dilution).
Immunofluorescent staining
W1-AA677, E1-AA685 and control MCBS1 mouse mast cells were grown on fibronectin-coated chamber slides and labeled with the appropriate mAb or isotype control as used for flow cytometry. A1 antibody was indirectly labeled with 9E10 anti-myc secondary mouse mAb and then RPE-labeled rabbit anti-mouse (Dako). Cells were counterstained with 4′,6′-diamidino-2 phenylindole (DAPI, Sigma, Gillingham, Dorset, UK) and the slide was mounted using fluorescent mounting medium. Cells were visualized using a computer imaging system (Cell F, Olympus, Germany).
Adhesion assays
Based on saturation of staining identified using flow cytometry, polyclonal pre- and post-immune rabbit sera were incubated with HLMC cells at a 1:10 dilution, and scFvs with HMC-1 and HLMCs at approximately 20 μg/ml for 30 min at room temperature. HLMCs and HMC-1 cell adhesion to BEAS-2B epithelial and primary HASMCs was then assessed as described previously [5, 6].
Immunoprecipitation of scFv-bound mast cell ligand
For immunoprecipitation experiments, anti-C-myc 9E10 was covalently coupled to protein A/G Agarose using the Pierce Crosslink Immunoprecipitation kit (Pierce) using the manufacturer’s instructions. ScFv A1 and E4 (80 μg) were then bound to 80 μl of 50 % (v/v) 9E10-proteinA/G agarose resin in 0.01 M sodium phosphate, 0.15 M NaCl; pH 7.2 for 16 h at 4 °C. Resin was washed 3 times in PBS and twice in lysis/wash buffer. HMC-1 membrane pellets were prepared as described above from 1.6 × 107 cells and then solubilised in 1.2 ml of lysis/wash buffer (0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1 % NP-40, 5 % glycerol, pH 7.4) by incubation on ice for 20 min. Samples were centrifuged (17000 g, 20 min, 4 °C) and supernatants collected. Pellets were resuspended in the same buffer and incubated and centrifuged as before. Supernatant was collected and pooled with the previously obtained supernatant. Soluble native HMC-1 membrane (400 μl) was applied to the scFv-9E10-protein A/G agarose resin and allowed to bind at RT for 5 h with rotating. In spin columns, the resin was centrifuged (800 g, 10 s), resin was then washed 4 times with 500 μl TBS and once with 200 μl of conditioning buffer (Pierce Crosslink Immunoprecipitation kit). Protein was then eluted in three 100 μl volumes of a low pH elution buffer (Pierce Crosslink Immunoprecipitation kit). Immunoprecipitated proteins were separated on SDS-PAGE gels and visualised by staining with Coommassie brilliant blue. The A1 specific band of interest was excised and analysed by in-gel trypsin digestion followed by peptide mass fingerprint using MALDI-ToF mass spectrometry (service run by the Protein Nucleic Acid Chemistry Laboratory, University of Leicester, UK).
Assessment of Kit phosphorylation in HMC-1
106 HMC-1 cells in 1 ml were treated with 20 μg/ml E4 or A1 antibody for 15 min at 37 °C, followed by 100 ng/ml SCF for 3 min at 37 °C; 30 mg/lane of protein extract was resolved in 4–12 % gradient SDS-PAGE gel transferred on 2 blots, probed with either pY99 [Santa Cruz, sc-7020], anti-Kit E1 [Santa Cruz, sc-17806] and beta-actin C4 [Santa Cruz, sc-47778].
Statistical analysis
Differences in adhesion between experimental conditions were examined using paired t tests where appropriate. p < 0.05 was considered statistically significant.
