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The synergistic effect of phytohemagglutinin and interferon-γ on the expression of tumor necrosis factor-α from RAW 264.7 cells
Immunology Letters 98 (2005) 137–143
The synergistic effect of phytohemagglutinin and interferon-␥ on the expression of tumor necrosis factor-␣ from RAW 264.7 cells Sung Ho Changa,b , Se Hwan Munc , Na Young Koa,b , Jun Ho Leea,b , Myung Ha Juna,b , Jin Young Seoa,b , Young Mi Kimd , Wahn Soo Choia,b , Erk Hera,b,∗ a
Department of Immunology, College of Medicine, Konkuk University, Danwol dong, Chungju 380-701, Republic of Korea b Bio-Food and Drug Research Center, Konkuk University, Chungju 380-701, Republic of Korea c Department of Allergy and Rheumatology, Asan Medical Center, Seoul, 138-736, Republic of Korea d Department of Pharmacy, Duksung Women’s University, Tobong-gu, 132-714, Republic of Korea Received 3 September 2004; received in revised form 28 October 2004; accepted 29 October 2004 Available online 15 December 2004
Abstract Tumor necrosis factor-␣ (TNF-␣) is a major cytokine of host immune reaction by foreign agents. Phytohemagglutinin (PHA) is a dynamic contributor to mitogenic stimulation and augmentation of host immune defense. Interferon-␥ (IFN-␥) induces induction of cytokines in macrophages and lymphocytes. The aim of this study was to examine the synergistic effects of PHA plus low dose IFN-␥ on TNF-␣ mRNA production, cytosolic levels, and secretion in RAW 264.7 cells. The cells were stimulated with PHA or IFN-␥ using various concentrations for various times. The effects of PHA on TNF-␣ expression appeared in dose- and time-dependent manners. The maximum doses of PHA and IFN␥ to produce them were 300 g/ml PHA and 10 ng/ml IFN-␥. The optimum time of PHA for the TNF-␣ mRNA production and release were 6 and 7 h after stimulation, respectively, whereas the time of IFN-␥ on them was achieved at 3 and 8 h. Although the TNF-␣ mRNA production, cytosolic levels, and secretion from the cells were slightly detected under 10 g/ml PHA and 1 ng/ml IFN-␥, the combination of PHA (10 g/ml) and IFN-␥ (1 ng/ml) greatly increased them, indicating the synergistic effect of PHA plus low dose IFN-␥ on TNF-␣ expression. © 2004 Elsevier B.V. All rights reserved. Keywords: Tumor necrosis factor-␣; Interferon-␥; Phytohemagglutinin; Synergistic effect
1. Introduction TNF-␣, also called cachectin, is a 17 kDa protein that is biologically active in the form of a compact trimer. It is produced mainly by monocytes and macrophages. TNF-␣ acts as an immunostimulant and is an important mediator of host resistance to many infectious agents and malignant tumors [1]. Macrophages were originally identified as the producer cells of TNF-␣ and are extensively used for isolation of TNF-␣ [2]. Lectins are proteins or glycoproteins that bind sugars specifically and agglutinate cells. They are widely distributed in nature, being found in animals, insects, plants, and microorganisms [3]. Several plant lectins, such as the PHA, ∗
Corresponding author. Tel.: +82 43 840 3769; fax: +82 43 851 9329. E-mail address: [email protected] (E. Her).
0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.10.029
concanavalin A (Con A), and pokeweed mitogen (PWM), are potent mitogens for T-lymphocytes [4]. The PHA could exert therapeutic effects due to its ability to assist remission and induction in certain malignancies, exhibit direct antitumor cytotoxic effects, reinforce responses against various infections, and exert these biological responses at effective dose levels, making it pharmaceutically cost-effective [5,6]. IFNs (IFN-␣, IFN-, and IFN-␥) display antiviral activity, antitumor activity, and impact cellular metabolism and differentiation [7,8]. IFN-␥ is considered as a crucial cytokine in modulating important functions of macrophages, including tumor cell cytotoxicity, antimicrobial activity, and increased killing of intracellular pathogens [9–11]. IFN-␥ is approved for treatment of the chronic granulomatous disease [12]. However, the IFN therapy is associated with significant toxicity, which can be divided into constitutional and hema-
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tologic effects [13–16]. Treatment with the IFN is always considered in the context of its significant side-effect profile. Thus, subsequent efforts have been made to evaluate the IFN together with other biological response modifiers, including IL-2 or other chemotherapeutic agents. However, these have been insufficient to make clear advantage [17,18]. The aim of this study was to know whether PHA might have a synergistic effect with a low dose of IFN-␥ to stimulate the TNF-␣ generation from macrophages. The synergistic effects on TNF-␣ mRNA expression, cytosolic levels, and secretion from mouse macrophage cell line RAW 264.7 cells were studied.
2. Materials and methods
polymerase (Takara, Otsu Shiga, Japan). The primer sequences were 5 -GCAGGTCTACTTTGGAGTCATTGC3 (forward) and 5 -CATTCGAGGCTCCAGTGAAT TCCAG-3 (reverse) for mouse TNF-␣ and 5 -GGAGAAGATCTGGCACCACACC-3 (forward) and 5 -CCTGCTTGCTGATCCACATCTGCTGG-3 (reverse) for mouse -actin. The -actin primer was used as an internal control. The expected RT-PCR product sizes were 286 bp for TNF-␣ and 840 bp for -actin. Samples of cDNA were amplified by PCR using a PTC-150 PCR system (MJ Research, San Francisco, CA, USA). The amplified products were analyzed on 1.5% agarose gels containing 0.1 g/ml ethidium bromide. Band intensities were densitometrically analyzed using Bio-Rad Quantity One program (Bio-Rad Laboratories, Hercules, CA, USA).
2.1. Cell cultures
2.4. Preparation of protein samples
Mouse macrophage cell line (RAW 264.7) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM l-glutamine (all purchased from Life Technologies, Grand Islands, NY, USA) at 37 ◦ C under 6% CO2 in humidified air. For the experiments, RAW 264.7 cells were preincubated in 0.1% bovine serum albumin (Sigma, St. Louis, MO, USA)-supplemented DMEM for 2 h. They were then stimulated with PHA (Sigma, St. Louis, MO, USA), IFN-␥ (8.43 U/ng; R&D Systems, Minneapolis, MN, USA), or both. Lipopolysaccharide (1 g/ml) (LPS; Sigma, St. Louis, MO, USA), known as a potent stimulant of TNF-␣ induction in macrophages, was used as positive control.
RAW 264.7 cells were treated with IFN-␥, PHA or both. The supernatants and whole cell lysates were then collected for the measurement of TNF-␣. After harvesting the supernatants, the cells were washed twice with phosphate-buffered saline (PBS). The whole cell lysates were prepared in lysis buffer containing 0.5 mM PMSF, 10 g/ml leupeptin, and 1 mM EGTA, 1% (v/v) -mercaptoethanol. The supernatants and whole cell lysates were used for immunoblotting analysis.
2.2. Extraction of total RNA Total cellular RNA was extracted from activated RAW 264.7 cells with TRIzol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s protocol. The RNA purity was determined by the ratio A260 /A280 (all samples between 1.6 and 2), and its integrity was confirmed by the existence of clear bands for 18S and 28S RNA after electrophoresis through 1% agarose gel. 2.3. Reverse transcription-polymerase chain reaction (RT-PCR) The cDNA was synthesized from total RNA (2 g) in a reaction mixture (Takara, Otsu Shiga, Japan) containing Rous associated virus-2 (RAV-2) reverse transcriptase (Takara, Otsu Shiga, Japan) and the reverse primer of mouse TNF-␣ for 1 h at 42 ◦ C. This was followed by 5 min at 94 ◦ C for denaturation of reverse transcriptase. The cDNA was amplified for 32 cycles (94 ◦ C for 60 s, 56 ◦ C for 30 s, and 72 ◦ C for 1 min) in a reaction mixture (Takara, Otsu Shiga, Japan) containing Ex Taq DNA
2.5. Immunoblotting The supernatants or whole cell lysates (20–40 g for each) were electrophoresed on 16% polyacrylamide gels using SDS-PAGE and the proteins were subsequently electroblotted onto polyvinylidene fluoride membrane (Schdeicher & Schuell, Keene, NH, USA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline including 0.1% Tween-20 (TBS-T) for 1 h at room temperature and washed with TBS-T. The membranes were then incubated overnight at 4 ◦ C with the rabbit anti-mouse TNF-␣ antibody (Chemicon International Inc., Temecula, CA, USA) diluted in 1:125 TBS-T. After being washed three times with TBS-T, the membrane was incubated with goat anti-rabbit HRP-conjugated IgG (Chemicon International Inc., Temecula, CA, USA) for 3 h at 4 ◦ C and was developed using an ECL system (Amersham Pharmacia Biotech, Clevland, OH, USA) according to the manufacturer’s instructions. Densitometric analysis was completed using Quantity One program (Bio-Rad Laboratories, Hercules, CA, USA). 2.6. Confocal microscopy For the detection of cytosolic TNF-␣, RAW 264.7 cells were grown on Lab Tek chamber slide (Nalgene Nunc International, Rochester, NY, USA) and then stimulated for 4 h with PHA (10 g/ml), IFN-␥ (1 ng/ml), or both. The cells were washed three times with PBS, fixed in 4% formaldehyde, washed with PBS, and permeabilized for
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5 min in phosphate-buffered saline (PBS) containing 0.1% Triton X-100. The permeabilized cells were blocked for 1 h with blocking buffer consisting of 10% goat serum in PBS. After blocking, the cells were incubated with rabbit anti-mouse TNF-␣ antibody (1:250 dilution; Chemicon International Inc., Temecula, CA, USA), followed by FITClabelled goat anti-rabbit IgG (second antibody) (1:12,800 dilution; Chemicon International Inc., Temecula, CA, USA) for 30 min at 37 ◦ C. The cells were then washed with PBS, mounted with mounting medium (DAKO Corporation, Carpinteria, CA, USA), and then cover-slipped. Confocal images were collected using a Bio-Rad MRC-1024 confocal microscope system (Bio-Rad Laboratories, Hercules, CA, USA) using LaserSharp MRC-1024 software (Version 3.2; Bio-Rad Laboratories, Hercules, CA, USA). Quantification of fluorescence intensity was completed with Quantity One program (Bio-Rad Laboratories, Hercules, CA, USA).
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Fig. 2. Time-dependent effects of PHA on TNF-␣ mRNA production (A) and TNF-␣ secretion (B) from RAW 264.7 cells. The cells (1 × 106 cells/well) were treated with 300 g/ml of PHA for 0–48 h. (A) RT-PCR profile of macrophage TNF-␣ and -actin mRNA productions. (B) Immunoblotting profile of macrophage TNF-␣ secretion. LPS and -actin were used as positive and internal controls, respectively. Data were representative of the triplicate of two independent experiments.
2.7. Statistical analysis Data were expressed as a percentage of values for stimulated cells with LPS (positive control) and analyzed using paired Student’s t-test or analysis of variance (ANOVA) as appropriate. Results were considered significant when P < 0.05.
3. Results 3.1. Dose- and time-dependent effect of PHA on TNF-α expression in RAW 264.7 cells To investigate the dose-dependent effects of PHA, the cells were treated with increasing concentration of 0–1000 g/ml of PHA for 8 h. PHA caused TNF-␣ mRNA production and TNF-␣ secretion in a dose-dependent manner (Fig. 1A and B). Based on these observations, 300 g/ml of PHA was used
Fig. 1. Dose-dependent effects of PHA on TNF-␣ mRNA production (A) and TNF-␣ secretion (B) from RAW 264.7 cells. The cells (1 × 106 cells/well) were treated with increasing concentration of 0–1000 g/ml of PHA for 8 h. (A) RT-PCR profile of macrophage TNF-␣ and -actin mRNA production s. (B) Immunoblotting profile of macrophage TNF-␣ secretion. LPS and -actin were used as positive and internal controls, respectively. Data were representative of the triplicate of three independent experiments.
to measure the time-dependent effect of PHA, and 10 g/ml of PHA was used to determine the synergistic effect of PHA plus IFN-␥ on them. To examine the time-dependent effect of PHA on TNF␣ expression, the cells were stimulated with 300 g/ml of PHA for 0–48 h. TNF-␣ mRNA expression was achieved to peak when cells were stimulated during 6 h (Fig. 2A). However, maximum amount of TNF-␣ secretion appeared 7 h after stimulation (Fig. 2B). Taken together, the optimum effective time of PHA on TNF-␣ mRNA expression and TNF-␣ secretion was at 6 and 7 h, respectively (Fig. 2A and B). 3.2. Dose- and time-dependent effects of IFN-γ on TNF-α expression in RAW 264.7 cells To investigate the dose-dependent effect of IFN-␥ on TNF␣ expression, the cells were stimulated with increasing concentration of 0–30 ng/ml of IFN-␥ for 8 h. IFN-␥ caused TNF-␣ mRNA production and TNF-␣ secretion in a dosedependent manner (Fig. 3A and B). Based on these data, 10 ng/ml of IFN-␥ was used to measure the time-dependent effect of IFN-␥, and 1 ng/ml of IFN-␥ was used to determine the synergistic effect of PHA plus IFN-␥ on TNF-␣ expression. To examine the time-dependent effects of IFN-␥, RAW 264.7 cells were stimulated with 10 ng/ml IFN-␥ for 0–48 h. The effective time of IFN-␥ on TNF-␣ mRNA expression was 2–3 h (Fig. 4A), whereas the effect on TNF-␣ secretion was achieved when the cells were stimulated during 8 h (Fig. 4B). 3.3. Synergistic effects of PHA plus IFN-γ on TNF-α mRNA expression and TNF-α secretion from RAW 264.7 cells To examine the synergistic effects of PHA plus IFN-␥ on TNF-␣ mRNA expression, the cells were activated with PHA,
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Fig. 3. Dose-dependent effects of IFN-␥ on TNF-␣ mRNA production (A) and TNF-␣ secretion (B) from RAW 264.7 cells. The cells (1 × 106 cells/well) were treated with increasing concentration of 0–30 ng/ml of IFN-␥ for 8 h. A: RT-PCR profile of macrophage TNF-␣ and -actin mRNA expressions. B: Immunoblotting profile of macrophage TNF-␣ secretion. LPS and -actin were used as positive and internal controls, respectively. Data were representative of the duplicate of three independent experiments.
IFN-␥, or both. PHA or IFN-␥ alone was treated for 6 and 3 h, respectively (Figs. 1–4). Co-treatment of the cells with PHA plus IFN-␥ were stimulated with IFN-␥ for 3 h after pretreatment with PHA for 3 h (Figs. 1–4). As a result, very low levels of TNF-␣ mRNA expression were detected in the cells treated with PHA (20 ± 5%) or IFN-␥ (17 ± 7%) alone. However, cotreatment of PHA and IFN-␥ synergistically induced TNF-␣
Fig. 4. Time-dependent effect of IFN-␥ on TNF-␣ mRNA production (A) and TNF-␣ secretion (B) from RAW 264.7 cells. The cells (1 × 106 cells/well) were treated with 10 ng/ml IFN-␥ for 0–48 h. A: RTPCR profile of macrophage TNF-␣ and -actin mRNA productions. B: Immunoblotting profile of macrophage TNF-␣ secretion. LPS and -actin were used as positive and internal controls, respectively. Data were representative of the triplicate of three independent experiments.
mRNA expression from the cells (98 ± 4%) (P < 0.01; IFN-␥ versus PHA + IFN-␥ or PHA versus PHA + IFN-␥, Fig. 5A and B). To determine the synergistic effect of PHA plus IFN-␥ on TNF-␣ secretion, the cells were stimulated with PHA or IFN-␥ for 7 h and 8 h, respectively (Figs. 1–4). As a result, the combination of PHA and IFN-␥ showed the synergistic effect on TNF-␣ secretion from the cells (54 ± 8%), compared to PHA (6 ± 3%) or IFN-␥ (8 ± 3%) alone (P < 0.01; IFN-␥
Fig. 5. The synergistic effects of PHA plus IFN-␥ on TNF-␣ mRNA production (A) and TNF-␣ secretion (C) from RAW 264.7 cells. To examine the synergistic effects of PHA plus IFN-␥ on TNF-␣ mRNA expression, the cells were activated with PHA (10 g/ml), IFN-␥ (1 ng/ml), or both. PHA (10 g/ml) or IFN-␥ (1 ng/ml) alone was treated for 6 and 3 h, respectively. Co-treatment of the cells with PHA plus IFN-␥ were stimulated with IFN-␥ (1 ng/ml) for 3 h after pretreatment with PHA (10 g/ml) for 3 h. TNF-␣ mRNA expression of each group determined by RT-PCR analysis using total RNA (A). To determine the synergistic effect of PHA (10 g/ml) plus IFN-␥ (1 ng/ml) on TNF-␣ secretion, the cells were stimulated with PHA (10 g/ml) for 7 h after pretreatment with IFN-␥ (1 ng/ml) for 1 h. PHA (10 g/ml) or IFN-␥ (1 ng/ml) alone was treated for 7 and 8 h, respectively. TNF-␣ secretion of each group was determined by immnoblotting analysis using supernatant (C). (B) and (D) Densitometric analyses of RT-PCR and immunoblotting, respectively. LPS and -actin were used as positive and internal controls, respectively. After densitometric analysis, data were expressed as a percentage of values for stimulated cells with LPS (maximum stimulus) and plotted as means ± S.E.M. of the triplicate of two independent experiments. **: P < 0.01 compared to PHA (10 g/ml) or IFN-␥ (1 ng/ml) alone.
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Fig. 6. Immunoblotting analysis (A) and confocal microscopy analysis (C) of the synergistic effect of PHA plus IFN-␥ on cytosolic TNF-␣ levels in RAW 264.7 cells. The cells were stimulated with PHA (10 g/ml), IFN-␥ (1 ng/ml), or both for 4 h. For immunoblotting analysis the cells were disrupted to prepare the whole cell lysates. For confocal microscopy analysis the cells were fixed, blocked, and incubated with rabbit anti-mouse TNF-␣ antibody (1:250 dilution), followed by FITC-labelled goat anti-rabbit IgG (1:12,800). Confocal images were collected using a Bio-Rad MRC-1024 confocal microscope. (B) and (D) Densitometric analyses of immunoblotting and confocal image, respectively. (C) (a) LPS (1 g/ml); (b) second antibody alone; (c) medium alone; (d) IFN-␥ (1 ng/ml); (e) PHA (10 g/ml); (f) IFN-␥ (1 ng/ml) + PHA (10 g/ml). LPS was used as a positive control. After densitometric analysis or fluorescence intensity quantification, data were expressed as a percentage of values for stimulated cells with LPS (maximal stimulus) and plotted as means ± S.E.M. of the triplicate of two independent experiments. **: P < 0.01 compared to PHA (10 g/ml) or IFN-␥ (1 ng/ml) alone.
versus PHA + IFN-␥ or PHA versus PHA + IFN-␥, Fig. 5C and D). 3.4. Synergistic effect of PHA plus IFN-γ on cytosolic TNF-α levels from RAW 264.7 cells In order to further support and confirm the synergistic effect of PHA plus IFN-␥, the cytosolic TNF-␣ levels were evaluated using immunoblotting analysis and confocal microscopy analysis. To determine the effective time of PHA and IFN-␥ on cytosolic TNF-␣ levels, the cells were stimulated with PHA (300 g/ml) and IFN-␥ (10 ng/ml) for 0–24 h. The whole cell lysates were prepared for the measurement of cytosolic TNF-␣ levels. Cytosolic TNF-␣ levels were increased up to 4 h, and then sharply declined. Thus, the optimum effective time of PHA and IFN-␥ on cytosolic TNF-␣ level was 4 h (data not shown). The combination of PHA and IFN-␥ did show a synergistic effect on cytosolic TNF␣ levels in the cells (48 ± 3%). On the other hand, PHA (15 ± 5%) or IFN-␥ (7 ± 3%) alone had little effect on the
TNF-␣ levels (P < 0.01; IFN-␥ versus PHA + IFN-␥ or PHA versus PHA + IFN-␥, Fig. 6A and B). In the confocal microscopy analysis, cells were treated with PHA, IFN-␥, or both for 4 h. As a result, no significant binding of FITC-labelled goat anti-rabbit IgG alone to the cells was seen (5 ± 2%; Fig. 6C(b) and D). The combination of PHA and IFN-␥ showed a synergistic effect on cytosolic TNF-␣ levels from the cells (89 ± 9%), compared to IFN-␥ (25 ± 7%) or PHA (24 ± 5%) alone (P < 0.01; IFN-␥ versus PHA + IFN-␥ or PHA versus PHA + IFN-␥, Fig. 6C and D).
4. Discussion Among cytokines, IFN-␥ directly or indirectly increases the activity of cytotoxic T-cells, macrophages, and NK cells, all of which play a role in the immune response in tumor cells [7]. During activation of macrophages, they produce TNF-␣, which acts as an immunostimulant and important mediator of host resistance to many infectious agents [1]. IFN-␥ shows
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some promise in the treatment of human cancer and immunodeficiency disease. However, in some cases, systemic administration of high levels of IFNs has led to serious and even life-threatening consequences [13]. PHA could also exert the therapeutic effects of a biological response modifier by virtue of its ability to exhibit tumoricidal effect [5]. Thus, we have investigated a synergistic effect of the combination of PHA and low levels of IFN-␥ on TNF-␣ expression in RAW 264.7 cells. Lectins bind sugars specifically and agglutinate cells [5]. Results from confocal microscopy assay showed that PHA agglutinated the cells, and even 10 g/ml of PHA showed an agglutination. PHA is potent mitogen for T-cells [4]. Whereas the effect of PHA on T-cells has been elucidated, any study to examine an effect of PHA on macrophages has not been performed yet. However, several researches finding functions of mistletoe lectin in macrophages have been done [19,20]. To approach the aim of present study, RAW 264.7 cells were used. The cells are well known to highly express TNF-␣. Results presented here showed that PHA triggered TNF-␣ mRNA generation and TNF-␣ release by the cells in a dosedependent manner. The data also showed that the high effective dose of PHA on TNF-␣ expression was 100–300 g/ml which might be toxic in T-cells. On the other hand, the effective dose of PHA on T-cells was reported in the range of 1–10 g/ml [21], which was negligible effective concentration used in present study. Taken together, these data indicated that macrophages might be less sensitive to PHA than T-cells. IFN-␥ produced by NK and T-cells during an infection shifts macrophages from a resting to an activated state and primes macrophages for antimicrobial activity [22]. Data obtained by this study showed that IFN-␥ activated macrophages expressed TNF-␣ mRNA and secreted TNF␣ in a dose-dependent manner. Since data presented here showed that IFN-␥ worked earlier than PHA for TNF-␣ mRNA production, but for the TNF-␣ secretion they needed almost the same time, further studies could be conducted to examine the signal transduction pathways manifested by both. It has been reported that low doses of IFN-␥ (5 U/ml) were used to examine the synergistic effect of water-soluble chitosan oligomers plus IFN-␥ for induction of nitric oxide synthesis in murine peritoneal macrophages [23]. Others studies have also shown that low dose of IFN-␥ (5 or 10 U/ml) have a synergistic effect with phorbol ester on inducible nitric oxide synthase in RAW 264.7 cells [24] and in microglial cells [25]. Result presented here showed that PHA (10 g/ml) or 1 ng/ml (8.43 U/ml) of IFN-␥ alone evoked only negligible TNF-␣ mRNA production, TNF-␣ cytosolic levels, and TNF-␣ release in the cells, whereas their combination greatly increased the three properties. In conclusion, this study demonstrated that PHA synergistically activated the cells for expression, generation, and secretion of TNF-␣ in the presence of IFN-␥. Possible mechanisms have been proposed by which immune regulators may enhance the ac-
tivity of nuclear factor- B, activator protein-1, and interferon regulatory factor-1 binding to the promoter regions of many cytokines [21,25]. Thus, further studies on the cooperative interaction of transcriptional factors among signal transduction pathways will provide important clues in the understanding of synergistic expression of TNF-␣ by PHA and IFN-␥ in macrophages.
Acknowledgments This study was supported by Konkuk University in 2002
References [1] Klimp AH, de Vries EG, Scherphof GL, Daemen T. A potential role of macrophage activation in the treatment of cancer. Crit Rev Oncol Hematol 2002;44(2):143–61. [2] Trinchieri G. Regulation of tumor necrosis factor production by monocyte-macrophages and lymphocytes. Immunol Res 1991;10: 89–103. [3] Sharon N, Goldstein IJ. Lectins: more than insecticides. Science 1998;282(5391):1049. [4] Ashraf MT, Khan RH. Mitogenic lectins. Med Sci Monit 2003; 9(11):R265–9. [5] Rudiger H, Gabius HJ. Plant lectins: occurrence, biochemistry, functions and applications. Glycoconjugate J 2001;18(8):589–613. [6] Mody R, Joshi S, Chaney W. Use of lectins as diagnostic and therapeutic tools for cancer. J Pharmacol Toxicol Methods 1995;33(1): 1–10. [7] Samuel CE. Antiviral actions of interferons. Clin Microbiol Rev 2001;14(4):778–809. [8] Parmar S, Platanias LC. Interferons: mechanisms of action and clinical applications. Curr Opin Oncol 2003;15(6):431–9. [9] Sen GC. Viruses and interferons. Annu Rev Microbiol 2001;55: 255–81. [10] Frese M, Schwarzle V, Barth K, Krieger N, Lohmann V, Mihm S, Haller O, Bartenschlager R. Interferon-gamma inhibits replication of subgenomic and genomic hepatitis C virus RNAs. Hepatology 2002;35(3):694–703. [11] Muller U, Steinhoff U, Reis LF, Hemmi S, Pavlovic J, Zinkernagel RM, Aguet M. Functional role of type I and type II interferons in antiviral defense. Science 1994;264(5167):1918–21. [12] Jackson SH, Miller GF, Segal BH, Mardiney III M, Domachowske JB, Gallin JI, Holland SM. IFN-gamma is effective in reducing infections in the mouse model of chronic granulomatous disease (CGD). J Interferon Cytokine Res 2001;21(8):567–73. [13] Borden EC, Parkinson D. A perspective on the clinical effectiveness and tolerance of interferon-alpha. Semin Oncol 1998;25(suppl 1): 3–8. [14] Rhodes J, Jones DH, Bleehen NM. Increased expression of human monocyte HLA-DR antigens and Fc gamma receptors in response to human interferon in vivo. Clin Exp Immunol 1983;53(3):739–43. [15] Volpe R. Immunoregulation in autoimmune thyroid disease. New Engl J Med 1987;316(1):44–6. [16] Weetman AP. Autoimmune thyroiditis: predisposition and pathogenesis. Clin Endocrinol 1992;36(4):307–23. [17] Boccardo F, Rubagotti A, Canobbio L, Galligioni E, Sorio R, Lucenti A, Cognetti F, Ruggeri E, Landonio G, Baiocchi C, Besana C, Citterio G, Rosa MD, Calabresi F. Interleukin-2, interferon-alpha and interleukin-2 plus interferon-alpha in renal cell carcinoma. A randomized phase II trial. Tumori 1998;84:534–9.
S.H. Chang et al. / Immunology Letters 98 (2005) 137–143 [18] Agarwala SS, Kirkwood JM. Interferons in melanoma. Curr Opin Oncol 1996;8:167–74. [19] Burger AM, Mengs U, Schuler JB, Fiebig HH. Anticancer activity of an aqueous mistletoe extract (AME) in syngeneic murine tumor models. Anticancer Res 2001;21(3B):1965–8. [20] Yoon TJ, Yoo YC, Kang TB, Song SK, Lee KB, Her E, Song KS, Kim JB. Antitumor activity of the Korean mistletoe lectin is attributed to activation of macrophages and NK cells. Arch Pharm Res 2003;26(10):861–7. [21] Staples KJ, Bergmann MW, Barnes PJ, Newton R. Evidence for post-transcriptional regulation of interleukin-5 by dexamethasone. Immunology 2003;109:527–35. [22] Gessani S, Belardelli F. IFN-gamma expression in macrophages and its possible biological significance. Cytokine Growth Factor Rev 1998;9(2):117–23.
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[23] Seo WG, Pae HO, Kim NY, Oh GS, Park IS, Kim YH, Kim YM, Lee YH, Jun CD, Chung HT. Synergistic cooperation between water-soluble chitosan oligomers and interferon-gamma for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages. Cancer Lett 2000;159:189– 95. [24] Momose I, Terashima M, Nakashima Y, Sakamoto M, Ishino H, Nabika T, Hosokawa Y, Tanigawa Y. Phorbol ester synergistically increases interferon regulatory factor-1 and inducible nitric oxide synthase induction in interferon-gamma-treated RAW 264.7 cells. Biochim Biophys Acta 2000;1498(1):19–31. [25] Han IO, Kim HS, Kim HC, Joe EH, Kim WK. Synergistic expression of inducible nitric oxide synthase by phorbol ester and interferongamma is mediated through NF-kappa B and ERK in microglial cells. J Neurosci Res 2003;73(5):659–69.
The synergistic effect of phytohemagglutinin and interferon-␥ on the expression of tumor necrosis factor-␣ from RAW 264.7 cells Sung Ho Changa,b , Se Hwan Munc , Na Young Koa,b , Jun Ho Leea,b , Myung Ha Juna,b , Jin Young Seoa,b , Young Mi Kimd , Wahn Soo Choia,b , Erk Hera,b,∗ a
Department of Immunology, College of Medicine, Konkuk University, Danwol dong, Chungju 380-701, Republic of Korea b Bio-Food and Drug Research Center, Konkuk University, Chungju 380-701, Republic of Korea c Department of Allergy and Rheumatology, Asan Medical Center, Seoul, 138-736, Republic of Korea d Department of Pharmacy, Duksung Women’s University, Tobong-gu, 132-714, Republic of Korea Received 3 September 2004; received in revised form 28 October 2004; accepted 29 October 2004 Available online 15 December 2004
Abstract Tumor necrosis factor-␣ (TNF-␣) is a major cytokine of host immune reaction by foreign agents. Phytohemagglutinin (PHA) is a dynamic contributor to mitogenic stimulation and augmentation of host immune defense. Interferon-␥ (IFN-␥) induces induction of cytokines in macrophages and lymphocytes. The aim of this study was to examine the synergistic effects of PHA plus low dose IFN-␥ on TNF-␣ mRNA production, cytosolic levels, and secretion in RAW 264.7 cells. The cells were stimulated with PHA or IFN-␥ using various concentrations for various times. The effects of PHA on TNF-␣ expression appeared in dose- and time-dependent manners. The maximum doses of PHA and IFN␥ to produce them were 300 g/ml PHA and 10 ng/ml IFN-␥. The optimum time of PHA for the TNF-␣ mRNA production and release were 6 and 7 h after stimulation, respectively, whereas the time of IFN-␥ on them was achieved at 3 and 8 h. Although the TNF-␣ mRNA production, cytosolic levels, and secretion from the cells were slightly detected under 10 g/ml PHA and 1 ng/ml IFN-␥, the combination of PHA (10 g/ml) and IFN-␥ (1 ng/ml) greatly increased them, indicating the synergistic effect of PHA plus low dose IFN-␥ on TNF-␣ expression. © 2004 Elsevier B.V. All rights reserved. Keywords: Tumor necrosis factor-␣; Interferon-␥; Phytohemagglutinin; Synergistic effect
1. Introduction TNF-␣, also called cachectin, is a 17 kDa protein that is biologically active in the form of a compact trimer. It is produced mainly by monocytes and macrophages. TNF-␣ acts as an immunostimulant and is an important mediator of host resistance to many infectious agents and malignant tumors [1]. Macrophages were originally identified as the producer cells of TNF-␣ and are extensively used for isolation of TNF-␣ [2]. Lectins are proteins or glycoproteins that bind sugars specifically and agglutinate cells. They are widely distributed in nature, being found in animals, insects, plants, and microorganisms [3]. Several plant lectins, such as the PHA, ∗
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0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.10.029
concanavalin A (Con A), and pokeweed mitogen (PWM), are potent mitogens for T-lymphocytes [4]. The PHA could exert therapeutic effects due to its ability to assist remission and induction in certain malignancies, exhibit direct antitumor cytotoxic effects, reinforce responses against various infections, and exert these biological responses at effective dose levels, making it pharmaceutically cost-effective [5,6]. IFNs (IFN-␣, IFN-, and IFN-␥) display antiviral activity, antitumor activity, and impact cellular metabolism and differentiation [7,8]. IFN-␥ is considered as a crucial cytokine in modulating important functions of macrophages, including tumor cell cytotoxicity, antimicrobial activity, and increased killing of intracellular pathogens [9–11]. IFN-␥ is approved for treatment of the chronic granulomatous disease [12]. However, the IFN therapy is associated with significant toxicity, which can be divided into constitutional and hema-
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tologic effects [13–16]. Treatment with the IFN is always considered in the context of its significant side-effect profile. Thus, subsequent efforts have been made to evaluate the IFN together with other biological response modifiers, including IL-2 or other chemotherapeutic agents. However, these have been insufficient to make clear advantage [17,18]. The aim of this study was to know whether PHA might have a synergistic effect with a low dose of IFN-␥ to stimulate the TNF-␣ generation from macrophages. The synergistic effects on TNF-␣ mRNA expression, cytosolic levels, and secretion from mouse macrophage cell line RAW 264.7 cells were studied.
2. Materials and methods
polymerase (Takara, Otsu Shiga, Japan). The primer sequences were 5 -GCAGGTCTACTTTGGAGTCATTGC3 (forward) and 5 -CATTCGAGGCTCCAGTGAAT TCCAG-3 (reverse) for mouse TNF-␣ and 5 -GGAGAAGATCTGGCACCACACC-3 (forward) and 5 -CCTGCTTGCTGATCCACATCTGCTGG-3 (reverse) for mouse -actin. The -actin primer was used as an internal control. The expected RT-PCR product sizes were 286 bp for TNF-␣ and 840 bp for -actin. Samples of cDNA were amplified by PCR using a PTC-150 PCR system (MJ Research, San Francisco, CA, USA). The amplified products were analyzed on 1.5% agarose gels containing 0.1 g/ml ethidium bromide. Band intensities were densitometrically analyzed using Bio-Rad Quantity One program (Bio-Rad Laboratories, Hercules, CA, USA).
2.1. Cell cultures
2.4. Preparation of protein samples
Mouse macrophage cell line (RAW 264.7) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM l-glutamine (all purchased from Life Technologies, Grand Islands, NY, USA) at 37 ◦ C under 6% CO2 in humidified air. For the experiments, RAW 264.7 cells were preincubated in 0.1% bovine serum albumin (Sigma, St. Louis, MO, USA)-supplemented DMEM for 2 h. They were then stimulated with PHA (Sigma, St. Louis, MO, USA), IFN-␥ (8.43 U/ng; R&D Systems, Minneapolis, MN, USA), or both. Lipopolysaccharide (1 g/ml) (LPS; Sigma, St. Louis, MO, USA), known as a potent stimulant of TNF-␣ induction in macrophages, was used as positive control.
RAW 264.7 cells were treated with IFN-␥, PHA or both. The supernatants and whole cell lysates were then collected for the measurement of TNF-␣. After harvesting the supernatants, the cells were washed twice with phosphate-buffered saline (PBS). The whole cell lysates were prepared in lysis buffer containing 0.5 mM PMSF, 10 g/ml leupeptin, and 1 mM EGTA, 1% (v/v) -mercaptoethanol. The supernatants and whole cell lysates were used for immunoblotting analysis.
2.2. Extraction of total RNA Total cellular RNA was extracted from activated RAW 264.7 cells with TRIzol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s protocol. The RNA purity was determined by the ratio A260 /A280 (all samples between 1.6 and 2), and its integrity was confirmed by the existence of clear bands for 18S and 28S RNA after electrophoresis through 1% agarose gel. 2.3. Reverse transcription-polymerase chain reaction (RT-PCR) The cDNA was synthesized from total RNA (2 g) in a reaction mixture (Takara, Otsu Shiga, Japan) containing Rous associated virus-2 (RAV-2) reverse transcriptase (Takara, Otsu Shiga, Japan) and the reverse primer of mouse TNF-␣ for 1 h at 42 ◦ C. This was followed by 5 min at 94 ◦ C for denaturation of reverse transcriptase. The cDNA was amplified for 32 cycles (94 ◦ C for 60 s, 56 ◦ C for 30 s, and 72 ◦ C for 1 min) in a reaction mixture (Takara, Otsu Shiga, Japan) containing Ex Taq DNA
2.5. Immunoblotting The supernatants or whole cell lysates (20–40 g for each) were electrophoresed on 16% polyacrylamide gels using SDS-PAGE and the proteins were subsequently electroblotted onto polyvinylidene fluoride membrane (Schdeicher & Schuell, Keene, NH, USA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline including 0.1% Tween-20 (TBS-T) for 1 h at room temperature and washed with TBS-T. The membranes were then incubated overnight at 4 ◦ C with the rabbit anti-mouse TNF-␣ antibody (Chemicon International Inc., Temecula, CA, USA) diluted in 1:125 TBS-T. After being washed three times with TBS-T, the membrane was incubated with goat anti-rabbit HRP-conjugated IgG (Chemicon International Inc., Temecula, CA, USA) for 3 h at 4 ◦ C and was developed using an ECL system (Amersham Pharmacia Biotech, Clevland, OH, USA) according to the manufacturer’s instructions. Densitometric analysis was completed using Quantity One program (Bio-Rad Laboratories, Hercules, CA, USA). 2.6. Confocal microscopy For the detection of cytosolic TNF-␣, RAW 264.7 cells were grown on Lab Tek chamber slide (Nalgene Nunc International, Rochester, NY, USA) and then stimulated for 4 h with PHA (10 g/ml), IFN-␥ (1 ng/ml), or both. The cells were washed three times with PBS, fixed in 4% formaldehyde, washed with PBS, and permeabilized for
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5 min in phosphate-buffered saline (PBS) containing 0.1% Triton X-100. The permeabilized cells were blocked for 1 h with blocking buffer consisting of 10% goat serum in PBS. After blocking, the cells were incubated with rabbit anti-mouse TNF-␣ antibody (1:250 dilution; Chemicon International Inc., Temecula, CA, USA), followed by FITClabelled goat anti-rabbit IgG (second antibody) (1:12,800 dilution; Chemicon International Inc., Temecula, CA, USA) for 30 min at 37 ◦ C. The cells were then washed with PBS, mounted with mounting medium (DAKO Corporation, Carpinteria, CA, USA), and then cover-slipped. Confocal images were collected using a Bio-Rad MRC-1024 confocal microscope system (Bio-Rad Laboratories, Hercules, CA, USA) using LaserSharp MRC-1024 software (Version 3.2; Bio-Rad Laboratories, Hercules, CA, USA). Quantification of fluorescence intensity was completed with Quantity One program (Bio-Rad Laboratories, Hercules, CA, USA).
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Fig. 2. Time-dependent effects of PHA on TNF-␣ mRNA production (A) and TNF-␣ secretion (B) from RAW 264.7 cells. The cells (1 × 106 cells/well) were treated with 300 g/ml of PHA for 0–48 h. (A) RT-PCR profile of macrophage TNF-␣ and -actin mRNA productions. (B) Immunoblotting profile of macrophage TNF-␣ secretion. LPS and -actin were used as positive and internal controls, respectively. Data were representative of the triplicate of two independent experiments.
2.7. Statistical analysis Data were expressed as a percentage of values for stimulated cells with LPS (positive control) and analyzed using paired Student’s t-test or analysis of variance (ANOVA) as appropriate. Results were considered significant when P < 0.05.
3. Results 3.1. Dose- and time-dependent effect of PHA on TNF-α expression in RAW 264.7 cells To investigate the dose-dependent effects of PHA, the cells were treated with increasing concentration of 0–1000 g/ml of PHA for 8 h. PHA caused TNF-␣ mRNA production and TNF-␣ secretion in a dose-dependent manner (Fig. 1A and B). Based on these observations, 300 g/ml of PHA was used
Fig. 1. Dose-dependent effects of PHA on TNF-␣ mRNA production (A) and TNF-␣ secretion (B) from RAW 264.7 cells. The cells (1 × 106 cells/well) were treated with increasing concentration of 0–1000 g/ml of PHA for 8 h. (A) RT-PCR profile of macrophage TNF-␣ and -actin mRNA production s. (B) Immunoblotting profile of macrophage TNF-␣ secretion. LPS and -actin were used as positive and internal controls, respectively. Data were representative of the triplicate of three independent experiments.
to measure the time-dependent effect of PHA, and 10 g/ml of PHA was used to determine the synergistic effect of PHA plus IFN-␥ on them. To examine the time-dependent effect of PHA on TNF␣ expression, the cells were stimulated with 300 g/ml of PHA for 0–48 h. TNF-␣ mRNA expression was achieved to peak when cells were stimulated during 6 h (Fig. 2A). However, maximum amount of TNF-␣ secretion appeared 7 h after stimulation (Fig. 2B). Taken together, the optimum effective time of PHA on TNF-␣ mRNA expression and TNF-␣ secretion was at 6 and 7 h, respectively (Fig. 2A and B). 3.2. Dose- and time-dependent effects of IFN-γ on TNF-α expression in RAW 264.7 cells To investigate the dose-dependent effect of IFN-␥ on TNF␣ expression, the cells were stimulated with increasing concentration of 0–30 ng/ml of IFN-␥ for 8 h. IFN-␥ caused TNF-␣ mRNA production and TNF-␣ secretion in a dosedependent manner (Fig. 3A and B). Based on these data, 10 ng/ml of IFN-␥ was used to measure the time-dependent effect of IFN-␥, and 1 ng/ml of IFN-␥ was used to determine the synergistic effect of PHA plus IFN-␥ on TNF-␣ expression. To examine the time-dependent effects of IFN-␥, RAW 264.7 cells were stimulated with 10 ng/ml IFN-␥ for 0–48 h. The effective time of IFN-␥ on TNF-␣ mRNA expression was 2–3 h (Fig. 4A), whereas the effect on TNF-␣ secretion was achieved when the cells were stimulated during 8 h (Fig. 4B). 3.3. Synergistic effects of PHA plus IFN-γ on TNF-α mRNA expression and TNF-α secretion from RAW 264.7 cells To examine the synergistic effects of PHA plus IFN-␥ on TNF-␣ mRNA expression, the cells were activated with PHA,
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Fig. 3. Dose-dependent effects of IFN-␥ on TNF-␣ mRNA production (A) and TNF-␣ secretion (B) from RAW 264.7 cells. The cells (1 × 106 cells/well) were treated with increasing concentration of 0–30 ng/ml of IFN-␥ for 8 h. A: RT-PCR profile of macrophage TNF-␣ and -actin mRNA expressions. B: Immunoblotting profile of macrophage TNF-␣ secretion. LPS and -actin were used as positive and internal controls, respectively. Data were representative of the duplicate of three independent experiments.
IFN-␥, or both. PHA or IFN-␥ alone was treated for 6 and 3 h, respectively (Figs. 1–4). Co-treatment of the cells with PHA plus IFN-␥ were stimulated with IFN-␥ for 3 h after pretreatment with PHA for 3 h (Figs. 1–4). As a result, very low levels of TNF-␣ mRNA expression were detected in the cells treated with PHA (20 ± 5%) or IFN-␥ (17 ± 7%) alone. However, cotreatment of PHA and IFN-␥ synergistically induced TNF-␣
Fig. 4. Time-dependent effect of IFN-␥ on TNF-␣ mRNA production (A) and TNF-␣ secretion (B) from RAW 264.7 cells. The cells (1 × 106 cells/well) were treated with 10 ng/ml IFN-␥ for 0–48 h. A: RTPCR profile of macrophage TNF-␣ and -actin mRNA productions. B: Immunoblotting profile of macrophage TNF-␣ secretion. LPS and -actin were used as positive and internal controls, respectively. Data were representative of the triplicate of three independent experiments.
mRNA expression from the cells (98 ± 4%) (P < 0.01; IFN-␥ versus PHA + IFN-␥ or PHA versus PHA + IFN-␥, Fig. 5A and B). To determine the synergistic effect of PHA plus IFN-␥ on TNF-␣ secretion, the cells were stimulated with PHA or IFN-␥ for 7 h and 8 h, respectively (Figs. 1–4). As a result, the combination of PHA and IFN-␥ showed the synergistic effect on TNF-␣ secretion from the cells (54 ± 8%), compared to PHA (6 ± 3%) or IFN-␥ (8 ± 3%) alone (P < 0.01; IFN-␥
Fig. 5. The synergistic effects of PHA plus IFN-␥ on TNF-␣ mRNA production (A) and TNF-␣ secretion (C) from RAW 264.7 cells. To examine the synergistic effects of PHA plus IFN-␥ on TNF-␣ mRNA expression, the cells were activated with PHA (10 g/ml), IFN-␥ (1 ng/ml), or both. PHA (10 g/ml) or IFN-␥ (1 ng/ml) alone was treated for 6 and 3 h, respectively. Co-treatment of the cells with PHA plus IFN-␥ were stimulated with IFN-␥ (1 ng/ml) for 3 h after pretreatment with PHA (10 g/ml) for 3 h. TNF-␣ mRNA expression of each group determined by RT-PCR analysis using total RNA (A). To determine the synergistic effect of PHA (10 g/ml) plus IFN-␥ (1 ng/ml) on TNF-␣ secretion, the cells were stimulated with PHA (10 g/ml) for 7 h after pretreatment with IFN-␥ (1 ng/ml) for 1 h. PHA (10 g/ml) or IFN-␥ (1 ng/ml) alone was treated for 7 and 8 h, respectively. TNF-␣ secretion of each group was determined by immnoblotting analysis using supernatant (C). (B) and (D) Densitometric analyses of RT-PCR and immunoblotting, respectively. LPS and -actin were used as positive and internal controls, respectively. After densitometric analysis, data were expressed as a percentage of values for stimulated cells with LPS (maximum stimulus) and plotted as means ± S.E.M. of the triplicate of two independent experiments. **: P < 0.01 compared to PHA (10 g/ml) or IFN-␥ (1 ng/ml) alone.
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Fig. 6. Immunoblotting analysis (A) and confocal microscopy analysis (C) of the synergistic effect of PHA plus IFN-␥ on cytosolic TNF-␣ levels in RAW 264.7 cells. The cells were stimulated with PHA (10 g/ml), IFN-␥ (1 ng/ml), or both for 4 h. For immunoblotting analysis the cells were disrupted to prepare the whole cell lysates. For confocal microscopy analysis the cells were fixed, blocked, and incubated with rabbit anti-mouse TNF-␣ antibody (1:250 dilution), followed by FITC-labelled goat anti-rabbit IgG (1:12,800). Confocal images were collected using a Bio-Rad MRC-1024 confocal microscope. (B) and (D) Densitometric analyses of immunoblotting and confocal image, respectively. (C) (a) LPS (1 g/ml); (b) second antibody alone; (c) medium alone; (d) IFN-␥ (1 ng/ml); (e) PHA (10 g/ml); (f) IFN-␥ (1 ng/ml) + PHA (10 g/ml). LPS was used as a positive control. After densitometric analysis or fluorescence intensity quantification, data were expressed as a percentage of values for stimulated cells with LPS (maximal stimulus) and plotted as means ± S.E.M. of the triplicate of two independent experiments. **: P < 0.01 compared to PHA (10 g/ml) or IFN-␥ (1 ng/ml) alone.
versus PHA + IFN-␥ or PHA versus PHA + IFN-␥, Fig. 5C and D). 3.4. Synergistic effect of PHA plus IFN-γ on cytosolic TNF-α levels from RAW 264.7 cells In order to further support and confirm the synergistic effect of PHA plus IFN-␥, the cytosolic TNF-␣ levels were evaluated using immunoblotting analysis and confocal microscopy analysis. To determine the effective time of PHA and IFN-␥ on cytosolic TNF-␣ levels, the cells were stimulated with PHA (300 g/ml) and IFN-␥ (10 ng/ml) for 0–24 h. The whole cell lysates were prepared for the measurement of cytosolic TNF-␣ levels. Cytosolic TNF-␣ levels were increased up to 4 h, and then sharply declined. Thus, the optimum effective time of PHA and IFN-␥ on cytosolic TNF-␣ level was 4 h (data not shown). The combination of PHA and IFN-␥ did show a synergistic effect on cytosolic TNF␣ levels in the cells (48 ± 3%). On the other hand, PHA (15 ± 5%) or IFN-␥ (7 ± 3%) alone had little effect on the
TNF-␣ levels (P < 0.01; IFN-␥ versus PHA + IFN-␥ or PHA versus PHA + IFN-␥, Fig. 6A and B). In the confocal microscopy analysis, cells were treated with PHA, IFN-␥, or both for 4 h. As a result, no significant binding of FITC-labelled goat anti-rabbit IgG alone to the cells was seen (5 ± 2%; Fig. 6C(b) and D). The combination of PHA and IFN-␥ showed a synergistic effect on cytosolic TNF-␣ levels from the cells (89 ± 9%), compared to IFN-␥ (25 ± 7%) or PHA (24 ± 5%) alone (P < 0.01; IFN-␥ versus PHA + IFN-␥ or PHA versus PHA + IFN-␥, Fig. 6C and D).
4. Discussion Among cytokines, IFN-␥ directly or indirectly increases the activity of cytotoxic T-cells, macrophages, and NK cells, all of which play a role in the immune response in tumor cells [7]. During activation of macrophages, they produce TNF-␣, which acts as an immunostimulant and important mediator of host resistance to many infectious agents [1]. IFN-␥ shows
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some promise in the treatment of human cancer and immunodeficiency disease. However, in some cases, systemic administration of high levels of IFNs has led to serious and even life-threatening consequences [13]. PHA could also exert the therapeutic effects of a biological response modifier by virtue of its ability to exhibit tumoricidal effect [5]. Thus, we have investigated a synergistic effect of the combination of PHA and low levels of IFN-␥ on TNF-␣ expression in RAW 264.7 cells. Lectins bind sugars specifically and agglutinate cells [5]. Results from confocal microscopy assay showed that PHA agglutinated the cells, and even 10 g/ml of PHA showed an agglutination. PHA is potent mitogen for T-cells [4]. Whereas the effect of PHA on T-cells has been elucidated, any study to examine an effect of PHA on macrophages has not been performed yet. However, several researches finding functions of mistletoe lectin in macrophages have been done [19,20]. To approach the aim of present study, RAW 264.7 cells were used. The cells are well known to highly express TNF-␣. Results presented here showed that PHA triggered TNF-␣ mRNA generation and TNF-␣ release by the cells in a dosedependent manner. The data also showed that the high effective dose of PHA on TNF-␣ expression was 100–300 g/ml which might be toxic in T-cells. On the other hand, the effective dose of PHA on T-cells was reported in the range of 1–10 g/ml [21], which was negligible effective concentration used in present study. Taken together, these data indicated that macrophages might be less sensitive to PHA than T-cells. IFN-␥ produced by NK and T-cells during an infection shifts macrophages from a resting to an activated state and primes macrophages for antimicrobial activity [22]. Data obtained by this study showed that IFN-␥ activated macrophages expressed TNF-␣ mRNA and secreted TNF␣ in a dose-dependent manner. Since data presented here showed that IFN-␥ worked earlier than PHA for TNF-␣ mRNA production, but for the TNF-␣ secretion they needed almost the same time, further studies could be conducted to examine the signal transduction pathways manifested by both. It has been reported that low doses of IFN-␥ (5 U/ml) were used to examine the synergistic effect of water-soluble chitosan oligomers plus IFN-␥ for induction of nitric oxide synthesis in murine peritoneal macrophages [23]. Others studies have also shown that low dose of IFN-␥ (5 or 10 U/ml) have a synergistic effect with phorbol ester on inducible nitric oxide synthase in RAW 264.7 cells [24] and in microglial cells [25]. Result presented here showed that PHA (10 g/ml) or 1 ng/ml (8.43 U/ml) of IFN-␥ alone evoked only negligible TNF-␣ mRNA production, TNF-␣ cytosolic levels, and TNF-␣ release in the cells, whereas their combination greatly increased the three properties. In conclusion, this study demonstrated that PHA synergistically activated the cells for expression, generation, and secretion of TNF-␣ in the presence of IFN-␥. Possible mechanisms have been proposed by which immune regulators may enhance the ac-
tivity of nuclear factor- B, activator protein-1, and interferon regulatory factor-1 binding to the promoter regions of many cytokines [21,25]. Thus, further studies on the cooperative interaction of transcriptional factors among signal transduction pathways will provide important clues in the understanding of synergistic expression of TNF-␣ by PHA and IFN-␥ in macrophages.
Acknowledgments This study was supported by Konkuk University in 2002
References [1] Klimp AH, de Vries EG, Scherphof GL, Daemen T. A potential role of macrophage activation in the treatment of cancer. Crit Rev Oncol Hematol 2002;44(2):143–61. [2] Trinchieri G. Regulation of tumor necrosis factor production by monocyte-macrophages and lymphocytes. Immunol Res 1991;10: 89–103. [3] Sharon N, Goldstein IJ. Lectins: more than insecticides. Science 1998;282(5391):1049. [4] Ashraf MT, Khan RH. Mitogenic lectins. Med Sci Monit 2003; 9(11):R265–9. [5] Rudiger H, Gabius HJ. Plant lectins: occurrence, biochemistry, functions and applications. Glycoconjugate J 2001;18(8):589–613. [6] Mody R, Joshi S, Chaney W. Use of lectins as diagnostic and therapeutic tools for cancer. J Pharmacol Toxicol Methods 1995;33(1): 1–10. [7] Samuel CE. Antiviral actions of interferons. Clin Microbiol Rev 2001;14(4):778–809. [8] Parmar S, Platanias LC. Interferons: mechanisms of action and clinical applications. Curr Opin Oncol 2003;15(6):431–9. [9] Sen GC. Viruses and interferons. Annu Rev Microbiol 2001;55: 255–81. [10] Frese M, Schwarzle V, Barth K, Krieger N, Lohmann V, Mihm S, Haller O, Bartenschlager R. Interferon-gamma inhibits replication of subgenomic and genomic hepatitis C virus RNAs. Hepatology 2002;35(3):694–703. [11] Muller U, Steinhoff U, Reis LF, Hemmi S, Pavlovic J, Zinkernagel RM, Aguet M. Functional role of type I and type II interferons in antiviral defense. Science 1994;264(5167):1918–21. [12] Jackson SH, Miller GF, Segal BH, Mardiney III M, Domachowske JB, Gallin JI, Holland SM. IFN-gamma is effective in reducing infections in the mouse model of chronic granulomatous disease (CGD). J Interferon Cytokine Res 2001;21(8):567–73. [13] Borden EC, Parkinson D. A perspective on the clinical effectiveness and tolerance of interferon-alpha. Semin Oncol 1998;25(suppl 1): 3–8. [14] Rhodes J, Jones DH, Bleehen NM. Increased expression of human monocyte HLA-DR antigens and Fc gamma receptors in response to human interferon in vivo. Clin Exp Immunol 1983;53(3):739–43. [15] Volpe R. Immunoregulation in autoimmune thyroid disease. New Engl J Med 1987;316(1):44–6. [16] Weetman AP. Autoimmune thyroiditis: predisposition and pathogenesis. Clin Endocrinol 1992;36(4):307–23. [17] Boccardo F, Rubagotti A, Canobbio L, Galligioni E, Sorio R, Lucenti A, Cognetti F, Ruggeri E, Landonio G, Baiocchi C, Besana C, Citterio G, Rosa MD, Calabresi F. Interleukin-2, interferon-alpha and interleukin-2 plus interferon-alpha in renal cell carcinoma. A randomized phase II trial. Tumori 1998;84:534–9.
S.H. Chang et al. / Immunology Letters 98 (2005) 137–143 [18] Agarwala SS, Kirkwood JM. Interferons in melanoma. Curr Opin Oncol 1996;8:167–74. [19] Burger AM, Mengs U, Schuler JB, Fiebig HH. Anticancer activity of an aqueous mistletoe extract (AME) in syngeneic murine tumor models. Anticancer Res 2001;21(3B):1965–8. [20] Yoon TJ, Yoo YC, Kang TB, Song SK, Lee KB, Her E, Song KS, Kim JB. Antitumor activity of the Korean mistletoe lectin is attributed to activation of macrophages and NK cells. Arch Pharm Res 2003;26(10):861–7. [21] Staples KJ, Bergmann MW, Barnes PJ, Newton R. Evidence for post-transcriptional regulation of interleukin-5 by dexamethasone. Immunology 2003;109:527–35. [22] Gessani S, Belardelli F. IFN-gamma expression in macrophages and its possible biological significance. Cytokine Growth Factor Rev 1998;9(2):117–23.
143
[23] Seo WG, Pae HO, Kim NY, Oh GS, Park IS, Kim YH, Kim YM, Lee YH, Jun CD, Chung HT. Synergistic cooperation between water-soluble chitosan oligomers and interferon-gamma for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages. Cancer Lett 2000;159:189– 95. [24] Momose I, Terashima M, Nakashima Y, Sakamoto M, Ishino H, Nabika T, Hosokawa Y, Tanigawa Y. Phorbol ester synergistically increases interferon regulatory factor-1 and inducible nitric oxide synthase induction in interferon-gamma-treated RAW 264.7 cells. Biochim Biophys Acta 2000;1498(1):19–31. [25] Han IO, Kim HS, Kim HC, Joe EH, Kim WK. Synergistic expression of inducible nitric oxide synthase by phorbol ester and interferongamma is mediated through NF-kappa B and ERK in microglial cells. J Neurosci Res 2003;73(5):659–69.