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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

CD172α and CD47 are members of glycoprotein expressed on macrophages and various immune cells, promote immune recognition and T cell stimulation that priming phagocytosis of pathogens and apoptotic bodies and malignant cell. Tumour-releasing immunosuppressive factor promotes tumour growth and transforms the tumour resident M1 phenotype of macrophage to M2 phenotype (TAMs) that promotes tumour progression by downregulating the expression of different surface receptor including CD172α and CD47. Recent studies have reported that CD172α and CD47 are involved in the pathogenesis and promote malignancies such as lymphoma, leukaemia, melanoma, lung cancer and multiple myeloma, and their expression varies during infection and malignancies. Autologous Hsp70 is well recognized for its role in activating macrophages leading to enhance production of inflammatory cytokines. It has been observed that Hsp70 derived from normal tissues do not elicit tumour immunity, while Hsp70 preparation from tumour cell was able to elicit tumour immunity. However, the role of exogenous autologous hsp70 on the formation of giant cells is completely unknown. Therefore, in the present study, we sought to investigate the effect of Hsp70–peptide complex on the expression of CD172α and CD47 receptors in normal peritoneal macrophages (NMO) and TAMs. Finding shows that the expression of CD172α and CD47 enhances in TAMs and it reverts back the suppressed function of TAMs into M1 state of immunoregulatory phenotype that promotes tumour regression by enhanced multinucleation and phagocytosis of malignant cells and significantly enhances the homotypic fusion of macrophages and polykaryon formation in vitro by enhancing the expression of SIRPα and IAP.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Macrophages are a fundamental pliable cell of the innate defence mechanisms of immune system, which can promote specific immunity by inducing T cell recruitment, activation and antigen presentation. Despite this, their presence within the tumour milieu, it has been associated with enhanced tumour progression and immunosuppression [1]. During infection and malignancies, it can undergo homotypic fusion to form polykaryon (MGC) or a giant cell which is considered as a hallmark of peritoneal macrophages. Formation of MGCs is believed to enhance the phagocytic function to limit the spread of infection. Giant cells are also important for tissue remodelling, wound healing and removal of even those pathogens which are non-phagocytosable due to increase in size. There are a number of surface molecules that are involved in the giant cell formation especially in homotypic fusion and phagocytosis of macrophages such as CD44, CD47, CD200, SIRP-α, IL-4R, E-cadherin, mannose receptor, and matrix metalloproteinases (MMPs) such as MMP-9 [2] and involve intracellular signal transduction. Signal regulatory protein-α (SIRP-α) also known as SHPS-1, SIRP-Α, etc. is a membrane glycoprotein belonging to the immunoglobulin (Ig) superfamily [3, 4] and is abundant in macrophages, dendritic cells, neutrophils [5-11] and plays an important role in cell–cell interaction, adherence to the substratum, and phagocytosis of tumour cell and apoptotic body. It has been reported that it might be involved in multinucleation. SIRP-α exhibits a negative regulatory function with CD47. The N-terminal extracellular region of this protein comprises three Ig-like domains, which associate with the ligand CD47 (integrin-associated protein (IAP)) to mediate specific biological functions [12] expressed on most of the immune cells including macrophages. It has been reported that, on immune cells, CD47 ligation by soluble monoclonal antibodies has been shown to inhibit cytokine production by antigen-presenting cells (APCs) and reduce interleukin-12 (IL-12) responsiveness by neonatal and adult T cells [13]. The interaction between CD47 and SIRP-α in macrophages is especially important for the regulation of phagocytosis of malignant cells [14]. Studies using CD47-deficient mice demonstrated that SIRP-α on MΦs recognizes CD47 as a marker of ‘self’ [15]. CD47–SIRP-α signalling prevents phagocytosis of normal haematopoietic cells by autologous macrophages and reduces the sensitivity of antibody- and complement-opsonized cells to phagocytosis. These results indicate that macrophages rely on CD47 expression to distinguish ‘self’ from ‘foreign’ and to set a threshold for macrophage-mediated phagocytosis of opsonized cells [16]. Several groups have recently shown that the CD47 expressed by leukaemia stem cells (LSCs) acts as an inhibitor of macrophage activity and directly promotes enhanced survival and engraftment of LSCs [17].

HSPs are conserved group of proteins expressed in almost all types of cells. There are different types of HSPs, and among all, hsp70 is highly conserved and immunogenic, capable of inducing antibody production and T cell activation. Hsp70 plays an important role in antigen presentation and cross-presentation of tumour antigen [18]. It can activate both normal peritoneal resident MΦs and tumour-associated macrophages (TAMs) leading to enhanced production of NO, H2O2, inflammatory cytokines, chemokines, and cytokine and chemokine receptors in addition to its profound role in eliciting cancer-specific immunity against the tumour by virtue of their ability to bind tumour-specific peptides. It has been observed that hsp70 preparations derived from normal tissues do not elicit tumour immunity, but it can activate macrophages [19-22].

Keeping the immunomodulatory function of hsp70 in mind, it is plausible to assume that the exogenous administration of tumour-derived hsp70 may increase the formation of macrophage polykaryon, in particular. Previous report has documented that CD172α and CD47 play important role in phagocytosis of apoptotic bodies and pathogens; CD47 act as an inhibitory role in phagocytosis with MΦs cells, and the role of exogenous autologous hsp70 on the formation of giant cells is completely lacking. Therefore, in the present study, we investigate the effect of tumour progression on the expression of CD172α and CD47, and the effect of tumour-derived hsp70–peptide complex has any role in the suppressed expression of CD172α and CD47 in multinucleation or giant cells formation in TAMs.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References
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.

Reagents

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 [25, 2]. 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 [2]. 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.

Cell viability

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.

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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. [25]. 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 [27] 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:

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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 [28]. 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.

Immunofluorescence

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 [28]. 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.

Flow cytometry

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 [29] 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.

Western blotting

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 [30]. The cytosolic proteins (6 μg/lane) were separated by 12% SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to PVDF membrane [31]. 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 isolation

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.

Reverse transcriptase-PCR

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 [32] 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
GenePrimerAmplicon lengthAnnealing temperatureNo. of cycle
CD172FP 5′-ATACGCAGACCTGAATGTGCCCAA-3′233–40072.1°C30
(SIRPαα)RP 5′-TGGCCACTCCATGTAGGACAAGAA-3′70.5°C30
CD47FP 5′-GAACTGCACTTCAGCAATG-3′180–35059.6°C30
(IAP)RP 5′-AACCTTCAGAAAGTCTGC-3′54.2°C30
β-actinFP 5′-ATC CAC GAA ACT ACC TTCAA-3′24458.7°C30
RP 5′-ATC CAC ACG GAG TAC TTG C-3′60.2°C30
Statistical analysis

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 [1] previously described. In each quantitative experiment with cells, 15 cells in total were analysed. A value of P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Characterization of autologous hsp70 and peritoneal macrophages

To determine whether purified peritoneal excaudate cells obtained from normal healthy and DL-bearing mice were macrophages, non-specific esterase staining of purified PECs was performed and positive red–brown colour formation (Fig. 1B) in more than 98% cells in homologous cell preparation was observed. Further, cells were characterized by CD14-PE staining which indicates that more than 98% cells are macrophages. Homologous preparations of hsp70 were purified from DL cell lysate in Sephadex column followed by DEAE-Sephacel column as described in Material and methods. The protein content of elute was measured by Bradford method, and 6 μg per lane of hsp70 preparations was separated by SDS-PAGE. The hsp70 purity is estimated to be greater than 95% in this preparation as determined by bands obtained in staining of SDS-PAGE of purified samples (Fig. 1A). The bands obtained were also checked and confirmed by immunoblots using hsp70-specific monoclonal antibodies.

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Figure 1. Characterization of hsp70 and macrophages. (A) DL cells were lysed in lysis buffer and purified from sephadex G 75 column followed by ADP/ATP agarose column. The protein concentration is estimated by standard method and 20 μg of proteins from elute were resolved on 12% polyacrylamide SDS gel, and bands were transferred to PVDF membrane and incubated with anti-mouse hsp70 antibody. Beta actin was used to show the equal loading of proteins, and (B) macrophages were harvested from both normal healthy and DL-bearing mice, adherence-purified and characterized by non-specific esterase staining, and (C) cells were double-stained with CD14-PE as a cell marker and isotype control-FITC to detect phenotype, green line was isotype control, Red TAMs and Black were Macrophages with 98.0 ± 2.0. Each experiments perform independently in triplicate.

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Hsp70 induces distinct morphological changes in macrophages

When adherence-purified macrophages from normal healthy and DL-bearing mice were treated with hsp70 isolated from DL cells, macrophages showed distinct morphological changes characterized by enhanced cytoplasmic extensions, compared with control group of macrophages incubated in medium only (Fig. 2A). Normal resident macrophages treated with hsp70 showed more prominent lamellipodeal and fillipodeal expression leading to oval shape of cells at the surface compared with TAMs (macrophages isolated from tumour-bearing host) at respective treatment of hsp70 and time of incubation. TAMs incubated in medium only were round in shape that can be compared with TAMs treated with hsp70 at the similar conditions. When normal resident macrophages and TAMs were treated with PMA at similar experimental conditions, corresponding observation was made as in the macrophages treated with hsp70. However, PMA treatment resulted in even more prominent morphological changes compared hsp70 treated TAMs. Interestingly, multinucleated giant cells were also observed in the cells treated with hsp70 or PMA. TAMs showed comparatively large number of cells undergoing fusion.

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Figure 2. Cells showing pattern of fusion in phenotype of macrophages. Upper panel shows normal peritoneal macrophages (NMO) while lower panel shows tumour-associated macrophages (TAMs) upon incubated in medium with or without PMA or hsp70; (B) shows numbers of multinucleate giant cells were counted, and fusion index was determined in both types of macrophages.

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Effect of autologous hsp70 on actin remodelling in macrophages

Morphological changes and fusion are directly correlated with actin remodelling which is taken place at the cell periphery. Therefore, next to understand the effect of hsp70 on actin remodelling and giant cell formation, macrophages were incubated in medium with or without PMA or hsp70 for different time periods of 6, 12 and 24 h, stained with phalloidin that stains F-actin and Hoechst 33258 that stains nucleus, and fluorescence microscopy was carried out. Normal resident macrophages incubated in medium only showed uniform polarization of F-actin at the cell periphery with single nucleus in the cell, whereas normal resident macrophages incubated in medium and activated with PMA showed polarization of F-actin at the cell periphery, densely stained at the several place. PMA treatment resulted in multinucleation of macrophages indicating that PMA induces macrophage homotypic fusion upon treatment. Hsp70 treatment to the normal resident macrophages showed corresponding actin polarization and nuclear staining, but more number of cells were observed undergoing fusion as compared to macrophages incubated in medium only or treated with PMA (Fig. 3). When TAMs were incubated in medium only, F-actin was found polarized at the several places; however, there was no sign of cell fusion as the cells were observed to contain single nucleus. However, PMA treatment resulted in corresponding F-actin polarization, but having tendency to fuse each other. Occasionally, multinucleation was observed in PMA-treated TAMs. Interestingly, when TAMs were treated with hsp70, F-actin polymerization was found to be enhanced with clear lamellipodeal expression and multinucleation. Hsp70 treatment resulted in greater extent of multinucleation or polykaryon formation in TAMs compared with normal resident macrophages treated with hsp70.

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Figure 3. Phalloidin and Hoechst 33258 staining of macrophages. Macrophages were isolated from normal healthy and DL-bearing mice and adherence-purified. Macrophages were incubated in medium with or without PMA or hsp70, and phalloidin staining for actin and Hoechst 33258 staining for nucleus were performed. In upper panel, A & B show the macrophages incubated with medium only, C & D show macrophages activated with PMA, and E–G show macrophages activated with hsp70. Similarly in lower panel, A & B show the macrophages incubated with medium only, C & D show macrophages activated with PMA, and E–G show macrophages activated with hsp70.

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Hsp70 induces macrophage polykaryon formation in time-dependent manner

As the fusion of macrophage and the formation of multinucleated cells is one of the hallmarks of chronic inflammation including in malignancies [1], we were interested in analysing the effect of autologous hsp70 on macrophage fusion. To this end, we stimulated the peritoneal macrophage with autologous hsp70 for different 6, 12 and 24 h of incubation and stained with haematoxylin and eosin. It was observed that normal resident macrophages incubated in medium and activated with hsp70 showed a large number of cells undergoing fusion compared with normal resident macrophages incubated in medium only, whereas TAMs treated with hsp70 showed comparatively less number of cells undergoing fusion compared with hsp70-treated normal resident macrophages (Fig. 2B). These observations were corresponding to the observation on the effect of hsp70 on actin remodelling.

Further, to know the effect of incubation of time on macrophage polykaryon formation, the cells were treated with hsp70 in medium and incubated for different time periods and number of nuclei per cell was analysed. It was found that on increasing the time periods of incubation, increased number of PMA-treated cells undergoing polykaryon formation compared with the normal resident macrophages incubated for increasing time periods in medium only (Table 2). Interestingly, fusion tendency of normal macrophages after the hsp70 treatment found to be more pronounced and progressively increased with increasing time course of incubation after treatment as observed by increasing fusion index. The fusion index in normal resident macrophages treated with hsp70 was 49%, 40%, 3% at 6 h, 80%, 70%, 40% at 12 h and 67%,

Table 2. Fusion index (FI) of macrophages showing multinucleate giant cells formation
 Number of nuclei per macrophage polykaryon
Time of incubation6 h12 h24 h
Conditions123451234512345
  1. Fusion Index (±FI%) was determined in ~1000 macrophages per observation.

NMO980000990000980000
NMO + PMA8020110078220068401000
NMO + hsp7060494031.5568070406557367386.9
TAMs970000980000970000
TAMs + PMA905000801030072207200
TAMs + Hsp7055453440598064255407072488

38%, 6.9% at 24 h was observed, while normal resident macrophages without stimulation showed 98% of macrophages were mononucleate. In contrast, TAMs incubated in medium only for increasing time; no polykaryon was formed as in the normal resident macrophages incubated in medium only. However, when TAMs treated with PMA showed formation of macrophage giant cells, but lesser extent to the normal resident macrophages treated with PMA at respective time of incubation. Hsp70 treatment to TAMs resulted in the progressive increase in the formation of giant cells corresponding to normal resident macrophages treated with hsp70.

Effect of Hsp70–peptide complex on the expression of fusion receptor by immunofluorescence

NMO and TAMs isolated from different groups of mice were incubated with medium alone or medium containing Hsp70, and expression of fusion receptor CD172α and CD47 was observed. It was observed that normal resident macrophages treated with Hsp70 resulted in significant increase in the expression of CD47 and CD172α receptors (R = 0.021 for CD47 and R = 0.027 for CD47) as compared to the normal resident macrophages of CD47 and CD172α incubated in medium only, which is corresponding to (P < 0.05) the observation that Hsp70 treatment resulted in increase in (Fig. 4A) the tendency of macrophages, while in Hsp70-mediated fusion receptor expression in TAMs, it was founded that TAMs treated with Hsp70 resulted in significant increase in the expression of CD47 and CD172α receptors as compared to the TAMs of CD47 and CD172α incubated (Fig. 5A) in medium alone, which is corresponding to (P < 0.05) the observation that hsp70 treatment resulted in increase in the tendency of TAMs.

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Figure 4. Effect of autologous Hsp70 of CD172α distribution in the macrophages. PECs harvested from normal mice and DL-bearing mice collected in RPMI supplemented with 10% FCS, wash thrice and both the macrophages were collected by adherence purification. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and double-stained with CD14-PE (Red) as a cell marker of macrophage, CD172α-FITC (Green) and Hoechst 33258 for nucleous (Blue). In (A) normal peritoneal macrophages (NMO) and in (B) TAMs upon incubation for colocalization of CD172α distribution and surface expression (CD172α) upon incubation. Averages of the colocalization coefficients in Pearson's correlation coefficient were calculated. For each experiment, 15 cells were analysed in 3 different optical regions. Each experiment performs in triplicates.

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image

Figure 5. Effect of autologous Hsp70 of CD47 distribution in the macrophages. PECs harvested from normal mice and DL-bearing mice collected in RPMI supplemented with 10% FCS, wash thrice and both the macrophages were collected by adherence purification. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and double-stained with CD14-PE (Red) as a cell marker of macrophage, CD47-FITC (Green) and Hoechst 33258 for nucleous (Blue). In (A) normal peritoneal macrophages (NMO) while (B) showing TAMs upon incubation for colocalization of CD47 distribution and surface expression (CD47) upon incubation. Averages of the colocalization coefficients in Pearson's correlation coefficient were calculated. For each experiment, 15 cells were analysed in 3 different optical regions. Each experiment performs in triplicates.

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Effect of Hsp70–peptide complex on the expression of fusion receptor by flow cytometry

Experiments were conducted to examine the effect of hsp70-mediated receptor expression in NMO and TAMs harvested from different groups of mice. Cells were incubated in medium and with and without PMA and hsp70 and the expression CD172α and CD47 was analyzed. It was founded that normal resident macrophages treated with Hsp70 resulted in significant increase in the expression of CD47 (149 ± 13.01) and CD172α (133 ± 9.13) receptors as compared to the normal resident macrophages of CD47 (68 ± 13.90) and CD172α (59 ± 7.03) incubated in (Fig. 4B) medium only, which is corresponding to (P < 0.05) the observation that Hsp70 treatment resulted in increase in the tendency of macrophages, while in Hsp70-mediated fusion receptor expression in TAMs, it was founded that TAMs treated with Hsp70 resulted in significant increase in the expression of (Fig. 5B) CD47 (49 ± 13.01) and CD172α (63 ± 9.13) receptors as compared to the TAMs of CD47 (8.09 ± 3.90) and CD172α (9.62 ± 4.03) incubated in medium alone, which is corresponding to (P < 0.05) the observation that hsp70 treatment resulted in increase in the tendency of TAMs.

Effect of Hsp70–peptide complex on the expression of fusion receptor by Western blotting

Western blot analysis of fusion receptor CD47 and CD172α expression in TAMs shows a band with an estimated molecular weight of 43–50 kDa and 37–50 kDa (known molecular mass of protein), respectively. Result shows that Hsp70 incubation increases the expression level of adhesion and (Figs. 6A and 7A) fusion protein in NMO and TAMs. TAMs without incubation (medium lone) show basal level of protein expression, while NMO with medium only shows sharp enhances expression a compare to TAMs.

image

Figure 6. CD172α expression enhances in macrophages. Peritoneal macrophages harvested from normal healthy mice treated with medium alone or medium containing hsp70 were recovered and after 24 h of incubation, purified fraction of cells were analysed by SDS-PAGE with marker and band were transfer to PVDF membrane, bands obtained were incubated with anti-CD172α-specific monoclonal antibody, and colour was developed as described in Materials and Methods. (A) showing the normal peritoneal macrophage and peritoneal tumour-associated macrophages harvested (TAMs) from tumour-bearing host. (B) showing kinetics of mRNA expression during incubation with hsp70 in both types of cells. The graph representative of three independent experiments in all of which both mRNAs shows as fold increase of mRNA over basel level. β- actin was used as an international control of RNA load.

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image

Figure 7. CD47 expression enhances in macrophages. Peritoneal macrophages harvested from normal healthy mice treated with medium alone or medium containing hsp70 were recovered and after 24 h of incubation, purified fraction of cells were analysed by SDS-PAGE with marker and band were transfer to PVDF membrane, bands obtained were incubated with anti-CD47 specific monoclonal antibody, and colour was developed as described in Materials and Methods. (A) showing the normal peritoneal macrophage and peritoneal tumour-associated macrophages harvested (TAMs) from tumour-bearing host. (B) showing kinetics of mRNA expression during incubation with hsp70 in both types of cells. The graph representative of three independent experiments in all of which both mRNAs shows as fold increase of mRNA over basel level. β- actin was used as an international control of RNA load.

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image

Figure 8. Putative model for correlating the modulation of MFR expression level with Hsp70 changes in TAMs. Cartoon showing the possible correlation with the effect of tumour progression on multinucleation and MFR expression on Mɸ and TAMs. Left panel showing that MFR plays a vital role in multinucleation, tumour-cell fusion and phagocytosis middle panel showing during impaired expression, all the function inhibited that promote tumour progression. While right panel showing the possible mechanism for the hsp70-mediated reversal of TAMs in to M1 phenotype of macrophages that significantly suppress tumour progression. *Many other molecules are also involved for the receptor expression, but this figure includes those molecules relevant to hsp70-induced signalling cascade.

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Effect of Hsp70–peptide complex on the expression of fusion receptor by RT-PCR

Experiments were conducted to examine the effect of hsp70-mediated fusion receptor expression in NMO and TAMs harvested from different groups of mice. Cells were incubated in medium and with and without PMA and Hsp70 and CD172α and CD47 gene expression was observed. It was founded that Hsp70-treated NMO and TAMs isolated from normal healthy host and DL-bearing mice to study the effect of fusion receptor CD172α and CD47 expression were observed. It was founded that normal resident macrophages treated with Hsp70 resulted in significant increase in the expression of CD47 (50%) and CD172α (20.4%) receptors as compared to the normal resident macrophages of CD47 and CD172α incubated in medium only (Control), which is corresponding to (P < 0.05) the observation that Hsp70 treatment resulted in increase in the tendency of macrophages, while in Hsp70-mediated fusion receptor (Figs. 6B and 7B) expression in TAMs, it was founded that TAMs treated with Hsp70 resulted in significant increase in the expression of CD47 (207%) and CD172α (176%) receptors as compared to the TAMs of CD47 and CD172α incubated in medium alone, which is corresponding to (P < 0.05) the observation that hsp70 treatment resulted in increase in the tendency of TAMs.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

In the present investigation, we show that exogenous administration of hsp70 derived from tumour cells triggers distinct morphological changes and enhances the homotypic fusion of both normal resident and tumour-associated macrophages leading to polykaryon formation. This observation is comparable to well-known role of hsp70 in macrophage activation that leads to the production of pro-inflammatory cytokines and chemokines in the macrophages. Contrary to this observation, PMA treatment results in the prominent morphological changes characterized by lamellipodeal and fillipodeal formations, but not induces homotypic fusion that may lead to multinucleated giant cell formation as compared to hsp70 treatment. Furthermore, the TAMs have significantly higher tendency to fuse and form multinucleated giant cells in time-dependent manner. It has been previously observed that tumour progression induces macrophage polykaryon formation [29, 33]. Moreover, when macrophages are incubated with tumour-cell-conditioned medium, they show increased tendency to form polykaryon [9]. Therefore, it can be assumed that tumour microenvironment switches the activation of signal cascade that might be implicated in enhanced tendency of TAMs to form macrophage polykaryon. However, there is no evidence suggesting the role of macrophage polykaryon in antitumour immunity in the host. Interestingly, fusion events have been proposed to play a possible role in cancer progression.

It has been shown that in vitro treatment of IL-4 to bone marrow and alveolar macrophage suspension culture inhibits the action of IL-3 and enhances the formation of macrophage polykaryon [1, 32]. We have previously reported that DL cell progression results in increased serum titre of anti-inflammatory cytokines IL-4, IL-10 and IL-13. Therefore, these cytokines can be attributed to enhanced property of tumour-associated macrophages to form multinucleated giant cell. Granulocyte–monocyte colony-stimulating factor (GM-CSF) [30, 6], monocyte colony-stimulating factor (M-CSF), transforming growth factor (TGF-β) and chemokines receptor activator of nuclear factor kappa-B ligand (RANKL) are other crucial factors that have been shown to induce multinucleation in the macrophage, which are also found increased in tumour microenvironment. In contrast, pro-inflammatory cytokines have been found to inhibit polykaryon formation of macrophages [31, 1, 33]. Additionally, it has been well established that phenotypes and functions of macrophages get suppressed and changed to M2 phenotype or type II polarized macrophages, which differ greatly in terms of receptor expression and cytokine and chemokine production [9, 34, 35]. Type II macrophages release immunoregulatory cytokines such as IL-4, IL-10 and IL-13 in lieu of pro-inflammatory cytokines such as IL-1, IL-12 and IL-15 [1, 6, 36]. Our result indicates that PMA activation of macrophages leads to prominent morphological changes as evident from scanning electron micrograph and fluorescent microscopy of actin staining, but exerts decreased capacity to form macrophage multikaryon. Therefore, it is possible that hsp70 treatment may either result in the production of anti-inflammatory cytokines or induce some other yet to be defined mechanism(s) leading to increased macrophage polykaryon formation.

Molecular mechanism leading to multinucleate giant cell formation at tumour microenvironment has not been completely worked out. It is evident that signal regulatory protein α (SIRPα), also known as CD172α which has immunoglobulin-like domain (Ig), interacts with CD47 on other macrophage and results in tethering or docking of macrophages to each other, signal inhibition and homotypic fusion [6, 37]. It is evident that the intracellular domains of the SIRP-α interacts with SHP-1 and SHP-2 through SH2-dependent interactions and regulates RTK signalling which triggers dephosphorylation events at cytoplasmic tail leading to inhibitory signal [6, 9] for growth and proliferation, but it has been shown that CD172α–CD47 interaction leads to the recruitment of other proteins to form fusion protein complex leading to macrophage homotypic fusion and subsequent macrophage polykaryon. However, it is not clear that only enhanced expression of SIRPα (CD172α) and CD47 on the cell surface (Table 3) of peritoneal macrophages results in increased instances of macrophages polykaryon or multinucleated giant cell formation and therefore it needs to be investigated in detail. In our experiments, hsp70 treatment results in significantly higher expression of CD172α as well as CD47 on the macrophages. Moreover, the expression of SIRPα and CD47 is higher in TAMs compared with normal resident macrophages treated with hsp70 which is corroborated with the previous observations demonstrating that some cytokines, in particular, IL-4, GM-CSF, M-CSF, IL-10 and TGF-β and chemokines such as RANKL trigger enhanced expression of SIRPα as well as CD47.

Table 3. CD172α and CD47 receptor expression on macrophage phenotypes
 MacrophagesTAMs
Medium only (Control)PMAHsp70Medium only (Control)PMAHsp70
  1. a

    ND, not defined; R, Pearson's correlation coefficient (+1 to −1); P = Unpaired Student's t-test <0.05.

Immunocytochemistry
Increases in mean value CD172α–X a 1.61X–X a 1.21X

R and P value

Med and Hsp70

 

R = 0.125

P = 0.001

  

R = 0.03

P = 0.005

 
Increases in mean value CD47–X 1.41X–X 1.10X

R and P value

Med and Hsp70

 

R = 0.045

P = 0.03

  

R = 0.45

P = 0.087

 
Flow cytometry
Increases in mean value CD172α–X (Control)1.70X2.89X–X2.4X3.20X

P value

Med and Hsp70

 P < 0.03  P < 0.04 
Increases in mean value CD47–X2.10X3.10X–X1.90X2.90X

P value

Med and Hsp70

 P = 0.05  P = 0.005 
Immunoblotting
Increases in mean value CD172α–X2.01X2.23X–X1.15X1.39X

P value

Med and Hsp70

 P = 0.009  P = 0.01 
Increases in mean value CD47–X a 2.49X–X a 2.10X

P value

Med and Hsp70

  a    a  
RT-PCR
Increases in mean value CD172α–X a 1.18X–X a 2.21X

P value

Med and Hsp70

 P = 0.005  P = 0.02 
Increases in mean value CD47–X 1.45X–X 1.32X

P value

Med and Hsp70

 P = 0.05  P = 0.002 

Therefore, the two most interesting findings of the present work are (a) the observation that murine Daltons lymphoma could decreased the expression profile of CD172α and CD47in M1 phenotype of macrophages that transform M1 phenotype into M2 or TAMs that negatively regulate tumor progression (Fig. 8) resulting in the inhibition of multinucleation, variety of functions including phagocytosis, signal transduction, site-specific gene expression, cell motility, wound healing, and inflammation and anti tumor immunologic responses against tumor progression while hsp70 peptide complex (b) incubation enhanced the suppressed expression CD172α and CD47 facilitate phagocytosis and able to revert back the suppressed phenotype of tumor associated macrophages or M2 phenotype to M1 functional state and on the other hand increases the instances of lamellipodia and cytoplasmic veils leading to enhanced adherence to the endothelium and antigen uptake.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

We are thankful to Dr S.S. Agarwal, IMS, BHU, Varanasi, for providing flow cytometry facility for the analysis of receptors, Dr O. N. Srivastava, Department of Physics, BHU, for SEM facility for analysis of morphology and Dr S. C. Lakhotia, Department of Zoology, BHU, for Immunoflurescence Imaging. We are grateful to UGC, New Delhi, for the student grants to PKG. This study is a part of his PhD Thesis.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.

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  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References
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