Animals and tumour model
Inbred populations of BALB/c (H2d) strain of mice of either sex were used at 8–12 weeks of age. All animals were kept in conventional cages (six animals in each cage) and received unsterilized food and water ad libitum. Experimental animals were inspected daily for survival. All animals were kept and maintained in utmost care under the guidelines of Animal Ethical Committee, Banaras Hindu University.
For tumour system, healthy mice of either sex at 8–12 weeks of age were injected intraperitoneally (i.p.) with 1.0 × 106 DL cells in 0.5 ml sterile PBS. The DL cells for transplantation were obtained from ascitic fluid of DL-bearing mice, where the yield of the cells is higher and maintained in ascitic form in vivo by serial transplantation.
RPMI 1640 culture medium was obtained from HiMedia, Mumbai, India. Foetal bovine serum (FBS) was obtained from Invitrogen, CA, USA, and goat serum was prepared and heat inactivated in the laboratory for plastic petriplate coating to purify TAMs. Anti-hsp70 was obtained from Biolegand, San Diego, CA, USA. CD47, CD172α conjugated with FITC and CD14 conjugated with PE from eBiosciences, San Diego, CA, USA. Goat IgG conjugated with alkaline phosphatase was obtained from Bangalore Genie, Banglore, India. The ATP/ADP agarose, G-75 sephadex column, phorbol 12-myristate 13-acetate (PMA), hoechst 33258 and phalloidin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Na2HPO4, KH2PO4, formaldehyde and trypsin were purchased from Qualigens, Mumbai, India, acetone was purchased from Rankem Ltd., Mumbai, India, and glutaraldehyde was obtained from Serva Electrophoresis, Heidelberg, Germany. All other chemicals otherwise stated were obtained from Qualigens.
Isolation and purification of hsp70
The hsp70 was isolated and purified as previously described [23, 24] with minor modifications. A total of 10 ml of packed DL cells, cultured in vitro in thermal stress condition, was homogenized in 40 ml of hypertonic buffer A without detergent (10 mm NaHCO3, 0.5 mm PMSF, pH 7.1) and centrifuged at 100,000 × g. The pellet was dissolved in buffer B (20 mm NaCl, 15 mm 2-ME, 3 mm MgCl2, 0.5 mm PMSF, pH 7.5) using Sephadex column (G-75). The elute was loaded on ADP or ATP-agarose column equilibrated with buffer B. The column was washed extensively with buffer B until protein was undetectable in elutes by absorbance at 280 nm. The buffer of elutes from ADP or ATP-agarose column was changed for buffer C. (20 mm Na2PO4, 20 mm NaCl, pH 7.0), and elutes were loaded onto DEAE-Sephacel column in buffer D (130 mm NaCl in buffer A). The supernatant was collected, and protein concentration was determined by Bradford method (Bradford, 1976). Protein was mixed with 6× loading dye and subjected to 12% SDS–PAGE, followed by wet electroblotting onto PVDF membrane. The membrane was blocked in 5% (w/v) fat-free skimmed milk, incubated overnight at 4 °C with anti-Hsp70 antibody (1:1000), washed in 1× PBS, incubated for 2 h at room temperature with ALP-conjugated goat anti-mouse IgG (1:1000). Finally, blots were incubated with nitro blue tetrazolium (NBT) reagent for 10 min at 37 °C for proper visualization of desired bands. For loading control, the same membrane was probed for β-actin with alkaline phosphatase-conjugated antibody (1:1000).
Purification and activation of macrophages
Macrophages were harvested from normal healthy and tumour-bearing mice by standard method [2, 25]. Briefly, mice were killed by cervical dislocation, and macrophages were harvested by peritoneal lavage as peritoneal exudates cells (PECs) using chilled serum-free culture medium RPMI-1640. PECs were cultured in Petri dishes (Tarson, Kolkata, India) at 37 °C in CO2 incubator (Shelab, Oregon, CA, USA) for 2 h. The culture was washed three times with lukewarm serum-free medium with gentle flushing to ensure that all DL and other non-adherent cells were removed and adherent cells were collected. Adherence-purified macrophages were seeded in 96-well flat-bottom culture plates (Tarson) at a cell density of 1.5 × 106 per well in the culture medium with or without PMA or hsp70 and incubated for time periods of 24 h.
Identification and characterization of macrophages
Adherent cells were washed in phosphate-buffered saline (PBS) and fixed for 1 min using 0.2% glutaraldehyde in PBS. The cells were stained for 1 h at pH 6.3 at room temperature with α-naphthyl butyrate as the substrate, with the addition of 36 mm NaF to control for the diffuse cytoplasmic staining that would not be attributable to monocytes . The reaction was stopped by removing the solution of α -naphthyl butyrate-NaF and rinsing the cells with water. Cells were counted by light microscopy (magnification × 400). Those cells that exhibited bright red, diffuse cytoplasmic staining were considered to be positive for non-specific esterase. Adherent cells were also fixed in 95% ethanol, stained with Giemsa and examined with a phase-contrast microscope. Further, macrophage purification was confirmed by flow cytometry analysis using CD14+ antibody conjugated with PE.
Peritoneal exudates cells (PECs) were harvested by peritoneal lavage using chilled serum-free culture medium RPMI 1640. The PECs were then transferred [2, 26] into a vented plastic tissue culture flask (Tarson) for culture at 37 °C in CO2 incubator (Shella, Oregon, CA, USA). The non-adherent cells were discarded by washing three times with lukewarm serum-free culture medium with gentle flushing. After incubation, control group viability of peritoneal macrophages was determined using exclusion by the Trypan Blue method. Trypan Blue (final concentration of 0.01% wt/vol; Sigma Chemical Co.) was added to each experimental group. Thereafter, aliquots of 10 μl were taken, and macrophages were counted in a hemocytometer chamber. Morphologic evaluation of viable cells (number of viable cells was multiplied by 100 and divided by the total number of cells) was performed (Data not shown) by light microscopy (Olympus CKX 41, Center Valley, PA, USA) at 430 original magnification. More than 99% cells were viable of adherence-purified macrophages before and upon incubation with and without autologous hsp70.
Macrophage cell-adhesion-spreading assay
Macrophages were subjected for spreading and attachment assay to examine their morphology, adhesion and fusion. Samples for scanning electron microscopy (SEM) were prepared according to the method of Tameike et al. . Normal peritoneal resident macrophages (NMO) and TAMs were fixed in 2.5% glutaraldehyde prepared in 0.1 m sterile PBS (137 mm Nacl, 8.1 mm Na2HPO4.12H2O, 2.68 mm KCl, 1.47 mm KH2PO4) for 30 min and post-fixed with 1% OsO4 overnight. After fixation, they were dehydrated using ethanol series. Samples were coated by gold-palladium (Quantum technology-SC7620) and observed using a scanning electron microscope (Zieas-EVO LS-10) at 25 kV at LV mode from central facility of Department of Zoology, BHU, Varanasi, India.
Macrophage giant cell formation and determination of the fusion index (FI)
Macrophage cell suspensions (1 × 106 cells/ml) were dispended as a 50 μl droplet in the centre of a well in 96-well flat-bottomed plate to produce a dense monolayer of NMO and TAMs separately and incubated for 6, 12 and 24 h at 37 °C under 5% CO2 in CO2 incubator. Cells were analysed for clustering and fusion after different time intervals using inverted microscope (Olympus CKX-41), and after that culture medium was removed from the well, cells were fixed and stained with haematoxylin and eosin and observed under microscope.
The fusion index rate of macrophages upon incubation with different condition and different time intervals was determined by  counting the number of stained nuclei in MGC and the total number of nuclei within a given field under a microscope at 100× magnification. The FI was calculated according to the following formula:
In each experiment, between 300 and 500 nuclei were counted from selected representative fields.
Hoechst 33258-phalloidin staining
Cytospin slides were prepared from normal peritoneal resident macrophage (NMO) and TAMs. Both groups of cells were incubated in medium with or without PMA and auto hsp70 for 6, 12 and 24 h of time intervals in RPMI 1640 containing 10% FCS, at 37 °C in 5% CO2 in humidified CO2 incubator. Cells were fixed in formaldehyde and permeabilized with PBS buffer containing 0.02% Triton X-100 and 4% formaldehyde . The fixed cells were stained with 10 mm/l of Hoechst 33258, a kind of blue fluorescent dye (excitation/emission maxima~350/461 nm), used commonly for labelling nuclei and 1 mm/ml of phalloidin (Sigma Chemical Co.) dye (excitation/emission maxima~350/461 nm) for cytoplasmic membranes (F-actin), respectively. After three washes with ice-cold sterile PBS, cells were mounted in DABCO. The images were visualized with a Nikon E800 upright fluorescence microscope (Melville, NY, USA) equipped with EXI aqua camera and NIS element software.
Cytospin slides were prepared from normal peritoneal resident macrophage (NMO) and TAMs. Both groups of cells were incubated in medium with or without PMA and auto hsp70 for 24 h of time in RPMI 1640 containing 10% FCS, at 37 °C in 5% CO2 in humidified CO2 incubator. Cells were fixed in 4% formaldehyde and permeabilized with PBS buffer containing 0.02% Triton X-100 . Then, anti-CD47 and CD172α-FITC antibody (1:100) were added, and incubation was continued overnight at 4 °C, followed by washing in PBS. The fixed cells were stained with 10 mM/ml of Hoechst 33258, a kind of blue fluorescent dye (excitation/emission maxima~350/461 nm) used commonly for labelling nuclei, respectively. After three washes with ice-cold sterile PBS, cells were mounted in DABCO. The images were visualized with a Nikon E800 upright fluorescence microscope equipped Hochest 33258 and FITC and PE filter with EXI aqua camera and NIS element software.
Cells were prepared from normal peritoneal resident macrophage (NMO) and TAMs. Both groups of cells were incubated in medium with or without PMA and auto hsp70 for 24 h of time in RPMI 1640 containing 10% FCS, at 37 °C in 5% CO2 in humidified CO2 incubator. Macrophages cells were suspended in complete media containing RPMI1640 with 10%FCS, 0.1%NaN3 and incubated  with anti-mouse CD47 (IAP), CD172α (SIRP-α) conjugated with FITC and anti-mouse CD14 conjugated with PE and isotype use as a positive control conjugated with FITC as per manufacturer's instruction. After washing, the cells were suspended in 0.1% PBS and 0.1%NaN3 and then analysed with a flow cytometry (BD Biosciences, Mountain View, CA, USA) equipped with an Innovate 90-5 (Coherent, Palo Alto, CA, USA) argon ion laser operating at 488 nm and 515 mw in light-regulated mode. Light scattering data and fluorescence parameter were collected by user-defined protocol and stored in list mode via lysis II program.
The protein lysate from cells of control and treated with PMA and Hsp70 was prepared in RIPA buffer and centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was collected, and protein concentration was determined by Bradford method . The cytosolic proteins (6 μg/lane) were separated by 12% SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to PVDF membrane . The membrane was blocked in 5% (w/v) fat-free skimmed milk, incubated for 2 h at 4 °C, washed in 1x PBS, incubated for overnight at 4 °C and immunoblotted with a mouse anti-CD47 and CD172α monoclonal antibody followed by incubation with alkaline phosphatase-conjugated antibody (Bangalore Genie, India) at a dilution of 1:5000.
RNA was extracted from the macrophages, and TAMs harvested from normal and DL-bearing mice of the control and experimental mice groups with TRI reagent as instructed by the manufacturer. High-quality RNA (as estimated by absorbance ratio A260/280P1.8) from different groups was resolved on 1% agarose formaldehyde gel and stained with ethidium bromide to check the integrity of 18S and 28S rRNA using UV transilluminator.
RNA (5 lg) from each group of mice was first reverse transcribed into cDNA using reverse transcriptase. The resulting cDNA was used as a template for PCR amplification using specific primers for CD172α, CD47 and β-actin as an internal control. Primer sequence and PCR conditions are mentioned in Table 1. A typical 20μl PCR  contained 20 mm ammonium sulphate, 75mm Tris-HCl, pH 8.8, 0.01% (vol/vol) Tween-20, 1 μm each primer, 2μl cDNA, 100 μm dNTPs (Finnzyme, Biolabs, New England), 0.1% (wt/vol) BSA and 0.25 U Taq polymerase (Genai, Banglore, India), and the following programme was used for reactions: 94 °C for 3 min, 30 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, 30 s using BioRad MJ mini thermal cycler (Hercules, CA, USA). PCR products were analysed by electrophoresis using a 2% (wt/vol) agarose gel stained with ethidium bromide, and the intensity of each band was measured under UV fluorescence using image analysis software from Gel documentation system (Bio-Red). The ratio of intensities of the bands for the gene product compared with the housekeeping gene β-actin was calculated and compared.
Table 1. Primer sequence and RT-PCR conditions of CD172α and CD47
|β-actin||FP 5′-ATC CAC GAA ACT ACC TTCAA-3′||244||58.7°C||30|
|RP 5′-ATC CAC ACG GAG TAC TTG C-3′||60.2°C||30|
Each value represents the mean SEM of three independent experiments in each group except for in vivo stimulation experiments where two independent experiments were conducted. Data are analysed using two-tailed Student's t-test on statistical software package Sigma Plot, version 12.0. Pearson's correlation coefficient was calculated for describing the colocalization correlation of the intensity distributions between two channels as  previously described. In each quantitative experiment with cells, 15 cells in total were analysed. A value of P < 0.05 was considered significant.