Biomedicine & Pharmacotherapy 68 (2014) 679–683
Available online at
ScienceDirect www.sciencedirect.com
Original article
Mononuclear cells phagocytic activity affects the crosstalk between immune and cancer cells Meir Djaldetti *, Hanna Bessler Laboratory for Immunology and Hematology Research, Rabin Medical Center, Hasharon Hospital, Petah Tiqva and the Sackler School of Medicine, Ramat Aviv, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 June 2014 Accepted 4 August 2014
The ‘‘professional phagocytes’’, i.e. monocytes and macrophages, play an important role as eliminators of pathogens and as essential components of the immune system. It is well established that monocytes induced for phagocytosis by various stimulators, produce cytokines that are closely related to inflammation. Considering the role of inflammation in carcinogenesis and the existence of an immune dialog between mononuclear and cancer cells, the aim of the present work was to examine cytokine production by immune cells stimulated for phagocytosis by latex particles and incubated with cells from HT-29 and RKO human cancer lines. Human peripheral blood mononuclear cells (PBMC) were incubated with various numbers of polysterene latex beads, 0.8 mm in diameter and the secretion of the following cytokines: TNF-a, IL-1b and IL-6, IL-10 and IL-1ra was examined before and after further incubation with cells of the both cancer lines. Phagocytosis of latex beads by PBMC caused an increased production of TNF-a, IL-1b and IL-10, whereas that of IL-6 declined. PBMC activated by latex beads and co-cultured with cancer cells generated lesser amount of the three pro-inflammatory cytokines TNF-a, IL-1b and IL6, while that of the anti-inflammatory IL-10 and IL-1ra remained unchanged. The results indicate that phagocytosis of polystyrene latex beads by human PBMC alters the dialogue between immune and cells of human colon carcinoma lines, an observation that may clarify the role of the immune cells in attenuating inflammation and restraining carcinogenesis. ß 2014 Published by Elsevier Masson SAS.
Keywords: Mononuclear cells Phagocytosis Cytokines Immunity Cancer cells
1. Introduction The phagocytic capacity of the peripheral blood mononuclear cells is an important component of the innate immune system. From morphological point of view, it depends in part on the cell membrane fluidity that permits a rapid change from a spherical to irregular shape, pursued by membrane ruffling, pseudopodia-like formations and finally engulfment of target particles. The ultrastructural alterations accompanying phagocytosis have been reviewed by Djaldetti et al. [1]. The engulfing activity of monocytes assigned as ‘‘professional phagocytes’’ may be activated by a large number of factors, acting as phagocytic stimulators, such as pathogens, latex beads [2], migration inhibitory factor (MIF) [3], zymosan particles [4], antibiotics [5], carbon nanotubes [6], intralipid [7,8], as well as lipopolysaccharide (LPS) and phorbol myristate acetase (PMA) [9,10]. Considering the role of phagocytes in the function of the immune system, the essential point to be
* Corresponding author. Laboratory for Immunology and Hematology Research, Rabin Medical Center, Hasharon Hospital, 7, Keren Kayemet Street, Petah Tiqva, Israel Tel.: +972 3 9372397; fax: +972 3 937238. E-mail addresses: [email protected], [email protected] (M. Djaldetti). http://dx.doi.org/10.1016/j.biopha.2014.08.004 0753-3322/ß 2014 Published by Elsevier Masson SAS.
stressed is that these cells are engaged not only in clearance and destroying pathogens by a number of molecular mechanisms, but they are also capable to produce cytokines linked with inflammatory processes throughout the phagocytic process. Thus, human mononuclear cells incubated with zymozane or latex particles produced one- or two-fold amounts of IL-8 [4]. Rat peritoneal macrophages expressed a marked increase in IL-1b secretion following phagocytosis of latex beads comparable to cells stimulated with LPS [11]. Monocytes activated for phagocytosis by antibiotics, such as erythromycin, mixifloxacin and doxycyclin showed a significantly increased production of IL-1b and IL-6 [5]. On the other hand, cytokines added to phagocytic cells may affect their engulfing ability. Thus, while TNF-a and granulocyte/macrophage colony stimulating factor enhanced microglial cells phagocytosis, IL-4 and transforming growth factor-beta exerted an inhibitory effect on that cell function [12]. In a previous study [13], we have reported that human peripheral blood mononuclear cells (PBMC) are vividly activated for cytokine production following incubation with cancer cells, creating an immune dialogue between these two types of cells. The released inflammatory cytokines play an important role in initiation, development and maintenance of chronic inflammation linked closely with carcinogenesis. Moreover,
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M. Djaldetti, H. Bessler / Biomedicine & Pharmacotherapy 68 (2014) 679–683
we have shown that the crosstalk between immune and cancer cells is affected by a number of drugs, nutrients and chemicals [14]. Following these observations, the question has been posed if phagocytosis of latex particles will affect the immune crosstalk between human PBMC and cells of two human colon cancer lines, expressed by a modulation of cytokine production. The aim of the study therefore, was to examine the production of the proinflammatory cytokines TNF-a, IL-1b, and IL-6, as well as the anti-inflammatory cytokines IL-10 and IL-1ra by PBMC incubated with latex beads and to assess potential modulation of the immune crosstalk between phagocytes and cells of HT-29 and RKO human cancer lines.
2. Materials and methods 2.1. Latex particles Polysterene latex beads, 0.8 mm in diameter, supplied as an aqueous suspension were used (Sigma, Israel). The number of latex particle/mL (n) was calculated using equation provided by the manufacturer. Latex beads’ suspension was diluted in 0.9% sodium chloride solution and 0.01 mL of various amounts of latex beads was added to 1.0 mL of cell cultures to get a final number of 108, 5 108, 109, and 5 109 particles/mL. 2.2. Cell preparation and culture conditions PBMC were separated from venous blood obtained from adult blood bank donors by gradient centrifugation using Lymphoprep1077 (Axis-Shield PoC AS, Oslo, Norway). The cells were washed twice in phosphate buffered saline (PBS) and suspended in RPMI1640 medium (Biological Industries, Beith Haemek, Israel) containing 1% penicillin, streptomycin and nystatin and supplemented with 10% fetal calf serum (designated as complete medium CM).
reach the final numbers indicated above. Cultures without latex served as controls. The cultures were maintained for 24 h at 37 8C in a humidified atmosphere containing 5% CO2. At the end of the incubation period, the cells and latex particles were removed by centrifugation at 12,000 rpm for 10 min, the supernatants were collected and kept at –70 8C until assayed for cytokine content. 2.6. Effect of phagocytosis on cytokine production by colon cancer cells An amount of 2 105 of HT-29 or RKO cells suspended in 1 mL of appropriate CM were incubated for 24 h at 37 8C in a humidified atmosphere containing 5% CO2 without or with latex particles at numbers as indicated above. At the end of the incubation period, the supernatants were collected and kept at –70 8C until assayed for cytokine content. 2.7. Cytokine content in the supernatants The concentration of cytokines in the supernatants was tested using ELISA kits specific for human cytokines (Biosource International, Camarillo, CA) as detailed in the guidelines provided by the manufacturer. Each kit is specific for one individual cytokine. The detection level of all cytokines was 30 pg/mL. The percentage of the CV of the ELISA assay for the cytokines examined was 2% to the mean or less. 2.8. Statistical analysis Data was analyzed using one way ANOVA with repeated measures to evaluate the effect of phagocytosis of latex particle on each cytokine production and two-tailed paired Student’s t-test to compare between the effect of the various numbers of latex particles and the control (incubated without latex). The results are expressed as mean SEM. P value of < 0.05 was considered as statistically significant.
2.3. Effect of latex phagocytosis on cytokine production 3. Results An amount of 2 106/ml of PBMC suspended in CM were incubated for 24 h without or with latex particles, at numbers as described above. The plates were incubated at 37 8C in a humidified atmosphere containing 5% CO2. At the end of the incubation period, the culture media were collected, the cells were removed by centrifugation and the supernatants were kept at – 70 8C until assayed for cytokine content.
Supernatants obtained from HT-29 or RKO cells incubated for 24 h without or with latex particles did not contain detectable amounts of any of the cytokines tested in the current study.
2.4. Cell lines
3.2. Effect of phagocytosis on IL-1b production
HT-29 and RKO human colon cancer cell lines were obtained from the American Type Cultural Collection, Rockville, MD. The cells were maintained in CM containing MacCoy’s 5A (Sigma, Israel) and modified eagle medium (MEM-Biological Industries Co, Beth-Haemek, Israel) respectively, supplemented with 10% FCS, 2 mM L-glutamine and antibiotics (penicillin, streptomycin and nystatin-Biological Industries Co, Beth-Haemek, Israel). The cells were grown in T-75 culture flasks (Nunc, Roskidle, Denmark) at 37 8C in a humidified atmosphere containing 5% CO2.
A dose-dependent stimulation of IL-1b secretion was observed when non-stimulated PBMC were incubated with increased number of latex beads (P = 0.0021) (Fig. 1). At doses of 5 108, 109 and 5 109 particles/mL, IL-1b secretion was enhanced by 24%, 43% and 90% respectively (P = 0.5, P = 0.027, and P = 0.0003, respectively). PBMC incubated with either HT-29 or RKO colon cancer cells produced IL-1b at amounts up to 5.5- and 3.3-fold higher, respectively. A dose-dependent inhibition of HT-29induced IL-1b production was found when increased doses of latex particles were added (P = 0.013). At 109 and 5 109 particles/ mL, IL-1b secretion by HT-29-stimulated PBMC was reduced by 17% and 48%, respectively (P = 0.069 and P = 0.0008, respectively). At lower numbers of latex particle (below 5 108/mL), the secretion of IL-1b by either non-stimulated or HT-29 stimulated PBMC was not affected. The addition of latex particles at any of the doses tested had no effect on RKO-induced IL-1b secretion by PBMC (P = 0.971).
2.5. Effect of phagocytosis on cytokine production by PBMC activated by cancer cells A 0.5 mL of PBMC (4 106/mL of CM) was incubated with 0.5 mL of CM or 0.5 mL of each type of cancer cells (4 105/mL suspended in the appropriate CM). Then, 0.01 mL of various latex particles suspensions was added at the onset of cultures to
3.1. Effect of phagocytosis on cytokine production by colon cancer cells
M. Djaldetti, H. Bessler / Biomedicine & Pharmacotherapy 68 (2014) 679–683
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Fig. 1. Effect of phagocytosis of latex beads on IL-1b secretion by non-stimulated PBMC (spontaneous) or induced by colon cancer cells (HT-29 and RKO). An amount of 2 106 PBMC were incubated for 24 h without or with 2 105 cancer cells in the absence or presence of latex beads at numbers as indicate. The level of IL-1b in the supernatants was detected by ELISA. Each column represents the mean of 15 experiments. Bars represent SEM. Asterisks represent statistically significant difference from cells incubated without latex beads (*P < 0.05; ***P < 0.001).
3.3. Effect of phagocytosis on TNFa production
3.4. Effect of phagocytosis on IL-6 production
Increased numbers of latex particles caused a dose-dependent enhancement of TNFa production by non-stimulated PBMC (P = 0.043) (Fig. 2). At 109 and 5 109 particles/mL TNFa secretion was increased by 27% and 56%, respectively (P = 0.012 and P = 0.0008, respectively). PBMC incubated with either HT-29 or RKO colon cancer cells produced higher amounts of TNFa by 5.4 and 4.6 times, respectively. Latex beads caused reduced secretion of TNFa by HT-29-stimulated PBMC (P = 0.037), but not by RKOstimulated PBMC (P = 0.733). At latex doses of 109 and 5 109 particles/mL, HT-29-induced TNFa production was inhibited by 15% and 31%, respectively (P = 0.059 and P = 0.0003, respectively). The production of TNFa by PBMC, non-stimulated or stimulated following contact with HT-29 was not affected by latex beads at numbers lower than 109 particles/mL. Latex particles added at the above mentioned doses exerted no significant effect on RKOinduced TNFa secretion by PBMC (P = 0.733), although at latex concentration of 5 109 particles/mL, the secretion of TNFa by PBMC induced by RKO cells was reduced by 16% (P = 0.0014).
A dose-dependent inhibited IL-6 production was observed when non-stimulated PBMC were incubated with increased number of latex particles (P = 0.01) (Fig. 3). At latex doses of 109 and 5x109 particles/ml, the production of IL-6 was reduced by 33% and 27.5% respectively (P = 0.04 and P = 0.002, respectively). IL-6 secretion was up to 1.5 and 1.37-fold higher following incubation of PBMC with HT-29 or RKO colon cancer cells, respectively. Addition of latex beads at numbers described above had no significant effect on IL-6 secretion induced by either HT-29 cells (P = 0.501) or RKO cells (P = 0.248). However, at 5 109 particles/ ml the secretion of IL-6 by PBMC induced by their incubation with HT-29 or RKO cells was reduced by 11.5% and 13%, respectively (P < 0.03). 3.5. Effect of phagocytosis on anti-inflammatory cytokine secretion The production of the anti-inflammatory cytokines IL-10 and IL1ra was not affected by incubation of non-stimulated PBMC with
Fig. 2. Effect of phagocytosis of latex beads on TNFa secretion by non-stimulated PBMC (spontaneous) or induced by colon cancer cells (HT-29 and RKO). An amount of 2 106 PBMC were incubated for 24 h without or with 2 105 cancer cells in the absence or presence of latex beads at numbers as indicated. The level of TNFa in the supernatants was detected by ELISA. Each column represents the mean of 15 experiments. Bars represent SEM. Asterisks represent statistically significant difference from cells incubated without latex beads (***P < < 0.001).
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Fig. 3. Effect of phagocytosis of latex beads on IL-6 secretion by non-stimulated PBMC (spontaneous) or induced by colon cancer cells (HT-29 and RKO). An amount of 2 106 PBMC were incubated for 24 hrs without or with 2 105 cancer cells in the absence or presence of latex beads at numbers as indicated. The level of IL-6 in the supernatants was detected by ELISA. Each column represents the mean of 15 experiments. Bars represent SEM. Asterisks represent statistically significant difference from cells incubated without latex beads (*P < < 0.05; ***P < < 0.001).
activated by phagocytosis of latex particles with cancer cells resulted in a reversal production of pro-inflammatory cytokines. This observation is of significant importance, since if this is the way that immune cells activated for phagocytosis act within the cancer environment in vivo, one can presume that mononuclear cells and macrophages infiltrating the tumor and its surroundings will be able to abate the inflammatory process by a decrease of proinflammatory cytokine production with a subsequent carcinogenesis restraint. It is of interest that PBMC activation following phagocytosis and contact with malignant cells is a dose-dependent phenomenon – the greater the number of both inert particles and malignant cells, the more the capacity for cytokine production is expressed. Given that in the present work, a complete washout of the latex beads from the incubation mixture following phagocytosis is practically unfeasible, one can argue that the observed modulation of the immune dialog between latex-activated PBMC and cancer cells may be due to a direct effect of latex particles on the tumor cells. However, this assumption might be declined, since incubation of cancer cells of both lines with latex particles did not have any effect on cytokine production in this study. It is notable that the type of cytokines produced by phagocyting cells depends not only on the promoter’s type, inducing the engulfment process, but also its size and chemical composition. Thus, human monocytes incubated with 20 nm carboxyl polystyrene particles stimulated IL-8 production, whereas 500 and 1,000 nm sized particles promoted both IL-6 and IL-8 secretion [15]. Based on our previous experience that 0.8 nm latex beads provided the best results as for the amount of phagocytized particles by each PBMC,
increased doses of latex beads as indicated in Table 1 (P = 0.176, P = 0.405, respectively). Incubation of PBMC with either HT-29 or RKO cells induced increased IL-10 production by 2.9 and 1.42 times, respectively; as for IL-1ra its secretion was enhanced by 1.4 and 1.3 times, respectively. The addition of latex beads at the four numbers tested in this study had no significant effect on either HT-29 or RKO-induced IL-10 production (P = 0.495, P = 0.488, respectively), or on HT-29 or RKO-induced IL-1ra generation (P = 0.684, P = 0.796, respectively). 4. Discussion In summing up the results of the present work, we have confirmed our previous observations that non-stimulated PBMC are able to produce a certain amount of TNF-a, IL-1b, IL-6, IL-1ra and IL-10 [13]. However, following their contact with cells of both colon cancer lines the level of these cytokines markedly increased, substantiating the existence of an immune relationship between mononuclear and cancer cells. Incubation of PBMC with latex beads induced an increased production of TNF-a, IL-1b and IL-10, whereas that of IL-6 declined. The remaining cytokines examined were not affected. On the other hand, when PBMC activated by latex phagocytosis were co-cultured with cancer cells, the generation of the three pro-inflammatory cytokines TNF-a, IL1b and IL-6 decreased, while that of the anti-inflammatory IL-10 and IL-1ra remained unaffected. It appears therefore, that the phagocytic process averted the crosstalk dialogue between immune and malignant cells, i.e. the contact between cells
Table 1 Effect of phagocytosis of latex particles on anti-inflammatory cytokine production. Latex beads/ml IL-10 (ng/mL) Spontaneous HT-29-induced RKO-induced IL-1ra (ng/mL) Spontaneous HT-29-induced RKO-induced
0
108
5 108
109
5 109
ANOVA
0.56 0.08 1.66 0.26 1.38 0.19
0.54 0.07 1.57 0.13 1.37 0.18
0.67 0.05 1.75 0.20 1.17 0.24
0.75 0.07 1.76 0.23 1.69 0.02
0.64 0.09 1.76 0.23 1.19 0.10
P = 0.176 P = 0.795 P = 0.483
971 119 1382 192 1267 233
883 47 1234 102 1265 130
948 123 1390 213 1105 353
970 121 1438 211 1301 245
1038 136 1366 196 1229 223
P = 0.405 P = 0.684 P = 0.796
An amount of 2 106 PBMC were incubated for 24 h without or with 2 105 cancer cells in the absence or presence of latex beads at numbers as indicated. The levels of IL-10 and IL-1ra were tested in the supernatants by ELISA. The results are expressed as mean SEM of 15 experiments.
M. Djaldetti, H. Bessler / Biomedicine & Pharmacotherapy 68 (2014) 679–683
we choose to carry out the experiments with only this particles’ size. Human monocytes and macrophages incubated with polystyrene or titania particles might differently affect the phagocyte response, as well as IL-1, IL-6, IL-8 and PGF2 generation, depending on the size and concentration of the particles [15–18]. Bacteria may modulate both phagocytic ability and cytokine production. Kang et al. [19] have shown the existence of a correlation between enhanced phagocytosis of Staphylococcus aureus by human mononuclear cells and IL-8 production. The question of how phagocytosis triggers cytokine production is intriguing. Apparently, the machinery activating inflammatory cytokine production in phagocyting macrophages depends on the factors promoting the engulfing process. Ketko et al. [20] have reported that surfactant protein A is able to directly bind flagellin via N-linked carbohydrate moieties with a subsequent augmentation of macrophage phagocytosis and IL-1b generation. A substantial number of studies have shown that Toll-like receptors (TLRs) play an important role in modulating various immune functions, including phagocytosis with enhanced cytokine production. These receptors are activated by lypopolysaccharides and lipoproteins existing on bacteria, such as S. aureus [19], or depressed by PRRs and PAMPs antagonists expressed in Pseudomonas aeruginosa [21]. Various antibiotics regulate phagocytosis and cytokine expression acting on different TLRs [6]. Hayashi et al. [22] have found that agonists of TLRs expressed on neutrophils induced an increase in their phagocytic ability, as well as cytokine production. Bearing in mind the relatively high number of TLRs in the phagocyting cells, it is conceivable that engulfed latex particles will activate these receptors with a subsequent cytokine production. In short, the present results indicate that phagocytosis of polystyrene latex beads by human peripheral blood mononuclear cells may modify the dialogue between immune and cells of HT-29 and RKO human colon carcinoma lines expressed by decreased production of the pro-inflammatory cytokines TNF-a, IL-1b and IL6 by the phagocyting cells. This observation may elucidate the beneficial role of the immune cells in abating inflammation and subsequently restraining carcinogenesis. References [1] Djaldetti M, Salman H, Bergman M, Djaldetti R, Bessler H. Phagocytosis – the mighty weapon of the silent warriors. Microsc Res Tech 2002;57:421–31. [2] Corradin SB, Buchmu¨ller-Rouiller Y, Maue¨l J. Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha. Eur J Immunol 1991;21:2553–8.
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[3] Onodera S, Suzuki K, Matsuno T, Kaneda K, Takagi M, Nishihira J. Macrophage migration inhibitory factor induces phagocytosis of foreign particles by macrophages in autocrine and paracrine fashion. Immunology 1997;92: 131–7. [4] Friedland JS, Constantin D, Shaw TC, Stylianou E. Regulation of interleukin-8 gene expression after phagocytosis of zymosan by human monocytic cells. J Leukoc Biol 2001;70:447–54. [5] Bode C, Diedrich B, Muenster S, Hentschel V, Weisheit C, Rommelsheim K, et al. Antibiotics regulate the immune response in both presence and absence of lipopolysaccharide through modulation of Toll-like receptors, cytokine production and phagocytosis in vitro. Int Immunopharmacol 2014;18:27–34. [6] Pondman KM, Sobik M, Nayak A, Tsolaki AG, Ja¨kel A, Flahaut E, et al. Complement activation by carbon nanotubes and its influence on the phagocytosis and cytokine response by macrophages. Nanomedicine 2014 [S1549-9634(14)00112-9]. [7] Szeinberg A, Messer G, Davidsohn J, Levanon M, Passwell JH. Studies on the effect of intralipid on human monocyte functions in vitro. Isr J Med Sci 1986;22:791–6. [8] Padeh S, Shor R, Passwell JH. Effect of intralipid on the phagocytic and microbicidal capacity of human monocytes in culture. J Pediatr Gastroenterol Nutr 1987;6:575–80. [9] Qu X, Cinar MU, Fan H, Pro¨ll M, Tesfaye D, Tholen E, et al. Comparison of the innate immune responses of porcine monocyte-derived dendritic cells and splenic dendritic cells stimulated with LPS. Innate Immun 2014 [Epub ahead of print]. [10] Repine JE, White JG, Clawson CC, Holmes BM. Effects of phorbol myristate acetate on the metabolism and ultrastructure of neutrophils in chronic granulomatous disease. J Clin Invest 1974;54:83–90. [11] Bergman M, Salman H, Bessler H, Omanski M, Punsky I, Djaldetti M. Interaction between phagocytosis and IL-1beta production by rat peritoneal macrophages. Biomed Pharmacother 2002;56:159–62. [12] von Zahn J, Mo¨ller T, Kettenmann H, Nolte C. Microglial phagocytosis is modulated by pro- and anti-inflammatory cytokines. Neuroreport 1997;8:3851–6. [13] Bessler H, Djaldetti M. Role of the equilibrium between colon cancer and mononuclear cells in cytokine production. Biomed Pharmacother 2010;64:706–11. [14] Djaldetti M, Bessler H. Modulators affecting the immune dialogue between human immune and colon cancer cells. World J Gastrointest Oncol 2014;6: 129–38. Review. [15] Prietl B, Meindl C, Roblegg E, Pieber TR, Lanzer G, Fro¨hlich E. Nano-sized and micro-sized polystyrene particles affect phagocyte function. Cell Biol Toxicol 2014;30:1–16. [16] Shanbhag AS, Jacobs JJ, Black J, Galante JO, Glant TT. Macrophage/particle interactions: effect of size, composition and surface area. J Biomed Mater Res 1994;28:81–90. [17] Gretzer C, Eriksson AS, Allde´n B, Ericson LE, Thomsen P. Monocyte activation on titanium-sputtered polystyrene surfaces in vitro: the effect of culture conditions on interleukin-1 release. Biomaterials 1996;17:851–8. [18] Adachi T, Takahara K, Taneo J, Uchiyama Y, Inaba K. Particle size of latex beads dictates IL-1b production mechanism. PLoS One 2013;8:e68499. [19] Kang HJ, Ha JM, Kim HS, Lee H, Kurokawa K, Lee BL. The role of phagocytosis in IL-8 production by human monocytes in response to lipoproteins on Staphylococcus aureus. Biochem Biophys Res Commun 2011;406:449–53. [20] Ketko AK, Lin C, Moore BB, LeVine AM. Surfactant protein A binds flagellin enhancing phagocytosis and IL-1b production. PLoS One 2013;8:e82680. [21] Lovewell RR, Patankar YR, Berwin B. mechanisms of phagocytosis and host clearance of Pseudomonas aeruginosa. Am J Physiol Cell Mom Physiol 2014;306:L591–603. [22] Hayashi F1, Means TK, Luster AD. Toll-like receptors stimulate human neutrophil function. Blood 2003;102:2660–9.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Mononuclear cells phagocytic activity affects the crosstalk between immune and cancer cells Meir Djaldetti *, Hanna Bessler Laboratory for Immunology and Hematology Research, Rabin Medical Center, Hasharon Hospital, Petah Tiqva and the Sackler School of Medicine, Ramat Aviv, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 June 2014 Accepted 4 August 2014
The ‘‘professional phagocytes’’, i.e. monocytes and macrophages, play an important role as eliminators of pathogens and as essential components of the immune system. It is well established that monocytes induced for phagocytosis by various stimulators, produce cytokines that are closely related to inflammation. Considering the role of inflammation in carcinogenesis and the existence of an immune dialog between mononuclear and cancer cells, the aim of the present work was to examine cytokine production by immune cells stimulated for phagocytosis by latex particles and incubated with cells from HT-29 and RKO human cancer lines. Human peripheral blood mononuclear cells (PBMC) were incubated with various numbers of polysterene latex beads, 0.8 mm in diameter and the secretion of the following cytokines: TNF-a, IL-1b and IL-6, IL-10 and IL-1ra was examined before and after further incubation with cells of the both cancer lines. Phagocytosis of latex beads by PBMC caused an increased production of TNF-a, IL-1b and IL-10, whereas that of IL-6 declined. PBMC activated by latex beads and co-cultured with cancer cells generated lesser amount of the three pro-inflammatory cytokines TNF-a, IL-1b and IL6, while that of the anti-inflammatory IL-10 and IL-1ra remained unchanged. The results indicate that phagocytosis of polystyrene latex beads by human PBMC alters the dialogue between immune and cells of human colon carcinoma lines, an observation that may clarify the role of the immune cells in attenuating inflammation and restraining carcinogenesis. ß 2014 Published by Elsevier Masson SAS.
Keywords: Mononuclear cells Phagocytosis Cytokines Immunity Cancer cells
1. Introduction The phagocytic capacity of the peripheral blood mononuclear cells is an important component of the innate immune system. From morphological point of view, it depends in part on the cell membrane fluidity that permits a rapid change from a spherical to irregular shape, pursued by membrane ruffling, pseudopodia-like formations and finally engulfment of target particles. The ultrastructural alterations accompanying phagocytosis have been reviewed by Djaldetti et al. [1]. The engulfing activity of monocytes assigned as ‘‘professional phagocytes’’ may be activated by a large number of factors, acting as phagocytic stimulators, such as pathogens, latex beads [2], migration inhibitory factor (MIF) [3], zymosan particles [4], antibiotics [5], carbon nanotubes [6], intralipid [7,8], as well as lipopolysaccharide (LPS) and phorbol myristate acetase (PMA) [9,10]. Considering the role of phagocytes in the function of the immune system, the essential point to be
* Corresponding author. Laboratory for Immunology and Hematology Research, Rabin Medical Center, Hasharon Hospital, 7, Keren Kayemet Street, Petah Tiqva, Israel Tel.: +972 3 9372397; fax: +972 3 937238. E-mail addresses: [email protected], [email protected] (M. Djaldetti). http://dx.doi.org/10.1016/j.biopha.2014.08.004 0753-3322/ß 2014 Published by Elsevier Masson SAS.
stressed is that these cells are engaged not only in clearance and destroying pathogens by a number of molecular mechanisms, but they are also capable to produce cytokines linked with inflammatory processes throughout the phagocytic process. Thus, human mononuclear cells incubated with zymozane or latex particles produced one- or two-fold amounts of IL-8 [4]. Rat peritoneal macrophages expressed a marked increase in IL-1b secretion following phagocytosis of latex beads comparable to cells stimulated with LPS [11]. Monocytes activated for phagocytosis by antibiotics, such as erythromycin, mixifloxacin and doxycyclin showed a significantly increased production of IL-1b and IL-6 [5]. On the other hand, cytokines added to phagocytic cells may affect their engulfing ability. Thus, while TNF-a and granulocyte/macrophage colony stimulating factor enhanced microglial cells phagocytosis, IL-4 and transforming growth factor-beta exerted an inhibitory effect on that cell function [12]. In a previous study [13], we have reported that human peripheral blood mononuclear cells (PBMC) are vividly activated for cytokine production following incubation with cancer cells, creating an immune dialogue between these two types of cells. The released inflammatory cytokines play an important role in initiation, development and maintenance of chronic inflammation linked closely with carcinogenesis. Moreover,
680
M. Djaldetti, H. Bessler / Biomedicine & Pharmacotherapy 68 (2014) 679–683
we have shown that the crosstalk between immune and cancer cells is affected by a number of drugs, nutrients and chemicals [14]. Following these observations, the question has been posed if phagocytosis of latex particles will affect the immune crosstalk between human PBMC and cells of two human colon cancer lines, expressed by a modulation of cytokine production. The aim of the study therefore, was to examine the production of the proinflammatory cytokines TNF-a, IL-1b, and IL-6, as well as the anti-inflammatory cytokines IL-10 and IL-1ra by PBMC incubated with latex beads and to assess potential modulation of the immune crosstalk between phagocytes and cells of HT-29 and RKO human cancer lines.
2. Materials and methods 2.1. Latex particles Polysterene latex beads, 0.8 mm in diameter, supplied as an aqueous suspension were used (Sigma, Israel). The number of latex particle/mL (n) was calculated using equation provided by the manufacturer. Latex beads’ suspension was diluted in 0.9% sodium chloride solution and 0.01 mL of various amounts of latex beads was added to 1.0 mL of cell cultures to get a final number of 108, 5 108, 109, and 5 109 particles/mL. 2.2. Cell preparation and culture conditions PBMC were separated from venous blood obtained from adult blood bank donors by gradient centrifugation using Lymphoprep1077 (Axis-Shield PoC AS, Oslo, Norway). The cells were washed twice in phosphate buffered saline (PBS) and suspended in RPMI1640 medium (Biological Industries, Beith Haemek, Israel) containing 1% penicillin, streptomycin and nystatin and supplemented with 10% fetal calf serum (designated as complete medium CM).
reach the final numbers indicated above. Cultures without latex served as controls. The cultures were maintained for 24 h at 37 8C in a humidified atmosphere containing 5% CO2. At the end of the incubation period, the cells and latex particles were removed by centrifugation at 12,000 rpm for 10 min, the supernatants were collected and kept at –70 8C until assayed for cytokine content. 2.6. Effect of phagocytosis on cytokine production by colon cancer cells An amount of 2 105 of HT-29 or RKO cells suspended in 1 mL of appropriate CM were incubated for 24 h at 37 8C in a humidified atmosphere containing 5% CO2 without or with latex particles at numbers as indicated above. At the end of the incubation period, the supernatants were collected and kept at –70 8C until assayed for cytokine content. 2.7. Cytokine content in the supernatants The concentration of cytokines in the supernatants was tested using ELISA kits specific for human cytokines (Biosource International, Camarillo, CA) as detailed in the guidelines provided by the manufacturer. Each kit is specific for one individual cytokine. The detection level of all cytokines was 30 pg/mL. The percentage of the CV of the ELISA assay for the cytokines examined was 2% to the mean or less. 2.8. Statistical analysis Data was analyzed using one way ANOVA with repeated measures to evaluate the effect of phagocytosis of latex particle on each cytokine production and two-tailed paired Student’s t-test to compare between the effect of the various numbers of latex particles and the control (incubated without latex). The results are expressed as mean SEM. P value of < 0.05 was considered as statistically significant.
2.3. Effect of latex phagocytosis on cytokine production 3. Results An amount of 2 106/ml of PBMC suspended in CM were incubated for 24 h without or with latex particles, at numbers as described above. The plates were incubated at 37 8C in a humidified atmosphere containing 5% CO2. At the end of the incubation period, the culture media were collected, the cells were removed by centrifugation and the supernatants were kept at – 70 8C until assayed for cytokine content.
Supernatants obtained from HT-29 or RKO cells incubated for 24 h without or with latex particles did not contain detectable amounts of any of the cytokines tested in the current study.
2.4. Cell lines
3.2. Effect of phagocytosis on IL-1b production
HT-29 and RKO human colon cancer cell lines were obtained from the American Type Cultural Collection, Rockville, MD. The cells were maintained in CM containing MacCoy’s 5A (Sigma, Israel) and modified eagle medium (MEM-Biological Industries Co, Beth-Haemek, Israel) respectively, supplemented with 10% FCS, 2 mM L-glutamine and antibiotics (penicillin, streptomycin and nystatin-Biological Industries Co, Beth-Haemek, Israel). The cells were grown in T-75 culture flasks (Nunc, Roskidle, Denmark) at 37 8C in a humidified atmosphere containing 5% CO2.
A dose-dependent stimulation of IL-1b secretion was observed when non-stimulated PBMC were incubated with increased number of latex beads (P = 0.0021) (Fig. 1). At doses of 5 108, 109 and 5 109 particles/mL, IL-1b secretion was enhanced by 24%, 43% and 90% respectively (P = 0.5, P = 0.027, and P = 0.0003, respectively). PBMC incubated with either HT-29 or RKO colon cancer cells produced IL-1b at amounts up to 5.5- and 3.3-fold higher, respectively. A dose-dependent inhibition of HT-29induced IL-1b production was found when increased doses of latex particles were added (P = 0.013). At 109 and 5 109 particles/ mL, IL-1b secretion by HT-29-stimulated PBMC was reduced by 17% and 48%, respectively (P = 0.069 and P = 0.0008, respectively). At lower numbers of latex particle (below 5 108/mL), the secretion of IL-1b by either non-stimulated or HT-29 stimulated PBMC was not affected. The addition of latex particles at any of the doses tested had no effect on RKO-induced IL-1b secretion by PBMC (P = 0.971).
2.5. Effect of phagocytosis on cytokine production by PBMC activated by cancer cells A 0.5 mL of PBMC (4 106/mL of CM) was incubated with 0.5 mL of CM or 0.5 mL of each type of cancer cells (4 105/mL suspended in the appropriate CM). Then, 0.01 mL of various latex particles suspensions was added at the onset of cultures to
3.1. Effect of phagocytosis on cytokine production by colon cancer cells
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Fig. 1. Effect of phagocytosis of latex beads on IL-1b secretion by non-stimulated PBMC (spontaneous) or induced by colon cancer cells (HT-29 and RKO). An amount of 2 106 PBMC were incubated for 24 h without or with 2 105 cancer cells in the absence or presence of latex beads at numbers as indicate. The level of IL-1b in the supernatants was detected by ELISA. Each column represents the mean of 15 experiments. Bars represent SEM. Asterisks represent statistically significant difference from cells incubated without latex beads (*P < 0.05; ***P < 0.001).
3.3. Effect of phagocytosis on TNFa production
3.4. Effect of phagocytosis on IL-6 production
Increased numbers of latex particles caused a dose-dependent enhancement of TNFa production by non-stimulated PBMC (P = 0.043) (Fig. 2). At 109 and 5 109 particles/mL TNFa secretion was increased by 27% and 56%, respectively (P = 0.012 and P = 0.0008, respectively). PBMC incubated with either HT-29 or RKO colon cancer cells produced higher amounts of TNFa by 5.4 and 4.6 times, respectively. Latex beads caused reduced secretion of TNFa by HT-29-stimulated PBMC (P = 0.037), but not by RKOstimulated PBMC (P = 0.733). At latex doses of 109 and 5 109 particles/mL, HT-29-induced TNFa production was inhibited by 15% and 31%, respectively (P = 0.059 and P = 0.0003, respectively). The production of TNFa by PBMC, non-stimulated or stimulated following contact with HT-29 was not affected by latex beads at numbers lower than 109 particles/mL. Latex particles added at the above mentioned doses exerted no significant effect on RKOinduced TNFa secretion by PBMC (P = 0.733), although at latex concentration of 5 109 particles/mL, the secretion of TNFa by PBMC induced by RKO cells was reduced by 16% (P = 0.0014).
A dose-dependent inhibited IL-6 production was observed when non-stimulated PBMC were incubated with increased number of latex particles (P = 0.01) (Fig. 3). At latex doses of 109 and 5x109 particles/ml, the production of IL-6 was reduced by 33% and 27.5% respectively (P = 0.04 and P = 0.002, respectively). IL-6 secretion was up to 1.5 and 1.37-fold higher following incubation of PBMC with HT-29 or RKO colon cancer cells, respectively. Addition of latex beads at numbers described above had no significant effect on IL-6 secretion induced by either HT-29 cells (P = 0.501) or RKO cells (P = 0.248). However, at 5 109 particles/ ml the secretion of IL-6 by PBMC induced by their incubation with HT-29 or RKO cells was reduced by 11.5% and 13%, respectively (P < 0.03). 3.5. Effect of phagocytosis on anti-inflammatory cytokine secretion The production of the anti-inflammatory cytokines IL-10 and IL1ra was not affected by incubation of non-stimulated PBMC with
Fig. 2. Effect of phagocytosis of latex beads on TNFa secretion by non-stimulated PBMC (spontaneous) or induced by colon cancer cells (HT-29 and RKO). An amount of 2 106 PBMC were incubated for 24 h without or with 2 105 cancer cells in the absence or presence of latex beads at numbers as indicated. The level of TNFa in the supernatants was detected by ELISA. Each column represents the mean of 15 experiments. Bars represent SEM. Asterisks represent statistically significant difference from cells incubated without latex beads (***P < < 0.001).
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M. Djaldetti, H. Bessler / Biomedicine & Pharmacotherapy 68 (2014) 679–683
Fig. 3. Effect of phagocytosis of latex beads on IL-6 secretion by non-stimulated PBMC (spontaneous) or induced by colon cancer cells (HT-29 and RKO). An amount of 2 106 PBMC were incubated for 24 hrs without or with 2 105 cancer cells in the absence or presence of latex beads at numbers as indicated. The level of IL-6 in the supernatants was detected by ELISA. Each column represents the mean of 15 experiments. Bars represent SEM. Asterisks represent statistically significant difference from cells incubated without latex beads (*P < < 0.05; ***P < < 0.001).
activated by phagocytosis of latex particles with cancer cells resulted in a reversal production of pro-inflammatory cytokines. This observation is of significant importance, since if this is the way that immune cells activated for phagocytosis act within the cancer environment in vivo, one can presume that mononuclear cells and macrophages infiltrating the tumor and its surroundings will be able to abate the inflammatory process by a decrease of proinflammatory cytokine production with a subsequent carcinogenesis restraint. It is of interest that PBMC activation following phagocytosis and contact with malignant cells is a dose-dependent phenomenon – the greater the number of both inert particles and malignant cells, the more the capacity for cytokine production is expressed. Given that in the present work, a complete washout of the latex beads from the incubation mixture following phagocytosis is practically unfeasible, one can argue that the observed modulation of the immune dialog between latex-activated PBMC and cancer cells may be due to a direct effect of latex particles on the tumor cells. However, this assumption might be declined, since incubation of cancer cells of both lines with latex particles did not have any effect on cytokine production in this study. It is notable that the type of cytokines produced by phagocyting cells depends not only on the promoter’s type, inducing the engulfment process, but also its size and chemical composition. Thus, human monocytes incubated with 20 nm carboxyl polystyrene particles stimulated IL-8 production, whereas 500 and 1,000 nm sized particles promoted both IL-6 and IL-8 secretion [15]. Based on our previous experience that 0.8 nm latex beads provided the best results as for the amount of phagocytized particles by each PBMC,
increased doses of latex beads as indicated in Table 1 (P = 0.176, P = 0.405, respectively). Incubation of PBMC with either HT-29 or RKO cells induced increased IL-10 production by 2.9 and 1.42 times, respectively; as for IL-1ra its secretion was enhanced by 1.4 and 1.3 times, respectively. The addition of latex beads at the four numbers tested in this study had no significant effect on either HT-29 or RKO-induced IL-10 production (P = 0.495, P = 0.488, respectively), or on HT-29 or RKO-induced IL-1ra generation (P = 0.684, P = 0.796, respectively). 4. Discussion In summing up the results of the present work, we have confirmed our previous observations that non-stimulated PBMC are able to produce a certain amount of TNF-a, IL-1b, IL-6, IL-1ra and IL-10 [13]. However, following their contact with cells of both colon cancer lines the level of these cytokines markedly increased, substantiating the existence of an immune relationship between mononuclear and cancer cells. Incubation of PBMC with latex beads induced an increased production of TNF-a, IL-1b and IL-10, whereas that of IL-6 declined. The remaining cytokines examined were not affected. On the other hand, when PBMC activated by latex phagocytosis were co-cultured with cancer cells, the generation of the three pro-inflammatory cytokines TNF-a, IL1b and IL-6 decreased, while that of the anti-inflammatory IL-10 and IL-1ra remained unaffected. It appears therefore, that the phagocytic process averted the crosstalk dialogue between immune and malignant cells, i.e. the contact between cells
Table 1 Effect of phagocytosis of latex particles on anti-inflammatory cytokine production. Latex beads/ml IL-10 (ng/mL) Spontaneous HT-29-induced RKO-induced IL-1ra (ng/mL) Spontaneous HT-29-induced RKO-induced
0
108
5 108
109
5 109
ANOVA
0.56 0.08 1.66 0.26 1.38 0.19
0.54 0.07 1.57 0.13 1.37 0.18
0.67 0.05 1.75 0.20 1.17 0.24
0.75 0.07 1.76 0.23 1.69 0.02
0.64 0.09 1.76 0.23 1.19 0.10
P = 0.176 P = 0.795 P = 0.483
971 119 1382 192 1267 233
883 47 1234 102 1265 130
948 123 1390 213 1105 353
970 121 1438 211 1301 245
1038 136 1366 196 1229 223
P = 0.405 P = 0.684 P = 0.796
An amount of 2 106 PBMC were incubated for 24 h without or with 2 105 cancer cells in the absence or presence of latex beads at numbers as indicated. The levels of IL-10 and IL-1ra were tested in the supernatants by ELISA. The results are expressed as mean SEM of 15 experiments.
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we choose to carry out the experiments with only this particles’ size. Human monocytes and macrophages incubated with polystyrene or titania particles might differently affect the phagocyte response, as well as IL-1, IL-6, IL-8 and PGF2 generation, depending on the size and concentration of the particles [15–18]. Bacteria may modulate both phagocytic ability and cytokine production. Kang et al. [19] have shown the existence of a correlation between enhanced phagocytosis of Staphylococcus aureus by human mononuclear cells and IL-8 production. The question of how phagocytosis triggers cytokine production is intriguing. Apparently, the machinery activating inflammatory cytokine production in phagocyting macrophages depends on the factors promoting the engulfing process. Ketko et al. [20] have reported that surfactant protein A is able to directly bind flagellin via N-linked carbohydrate moieties with a subsequent augmentation of macrophage phagocytosis and IL-1b generation. A substantial number of studies have shown that Toll-like receptors (TLRs) play an important role in modulating various immune functions, including phagocytosis with enhanced cytokine production. These receptors are activated by lypopolysaccharides and lipoproteins existing on bacteria, such as S. aureus [19], or depressed by PRRs and PAMPs antagonists expressed in Pseudomonas aeruginosa [21]. Various antibiotics regulate phagocytosis and cytokine expression acting on different TLRs [6]. Hayashi et al. [22] have found that agonists of TLRs expressed on neutrophils induced an increase in their phagocytic ability, as well as cytokine production. Bearing in mind the relatively high number of TLRs in the phagocyting cells, it is conceivable that engulfed latex particles will activate these receptors with a subsequent cytokine production. In short, the present results indicate that phagocytosis of polystyrene latex beads by human peripheral blood mononuclear cells may modify the dialogue between immune and cells of HT-29 and RKO human colon carcinoma lines expressed by decreased production of the pro-inflammatory cytokines TNF-a, IL-1b and IL6 by the phagocyting cells. This observation may elucidate the beneficial role of the immune cells in abating inflammation and subsequently restraining carcinogenesis. References [1] Djaldetti M, Salman H, Bergman M, Djaldetti R, Bessler H. Phagocytosis – the mighty weapon of the silent warriors. Microsc Res Tech 2002;57:421–31. [2] Corradin SB, Buchmu¨ller-Rouiller Y, Maue¨l J. Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha. Eur J Immunol 1991;21:2553–8.
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