Biological Trace Element Research

, Volume 135, Issue 1, pp 334–344

In Vitro Effects of Calcium Fructoborate upon Production of Inflammatory Mediators by LPS-stimulated RAW 264.7 Macrophages

Authors

    • Department of BiochemistryUniversity of Craiova
  • Cristina Ciofrangeanu
    • Department of Biochemistry and Molecular BiologyUniversity of Bucharest
  • Raluca Ion
    • Department of Biochemistry and Molecular BiologyUniversity of Bucharest
  • Anisoara Cimpean
    • Department of Biochemistry and Molecular BiologyUniversity of Bucharest
  • Bianca Galateanu
    • Department of Biochemistry and Molecular BiologyUniversity of Bucharest
  • Valentina Mitran
    • Department of Biochemistry and Molecular BiologyUniversity of Bucharest
  • Dana Iordachescu
    • Department of Biochemistry and Molecular BiologyUniversity of Bucharest
Article

DOI: 10.1007/s12011-009-8488-5

Cite this article as:
Scorei, R.I., Ciofrangeanu, C., Ion, R. et al. Biol Trace Elem Res (2010) 135: 334. doi:10.1007/s12011-009-8488-5

Abstract

The present study is supported by our previous findings suggesting that calcium fructoborate (CF) has anti-inflammatory and antioxidant properties. Thus, we investigated the effects of CF on a model for studying inflammatory disorders in vitro represented by lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. This investigation was performed by analyzing the levels of some mediators released during the inflammatory process: cytokines such as tumor necrosis factor-α (TNF-α), interleukins IL-1β and IL-6 as well as cyclooxygenase-2 (COX-2), the main enzyme responsible for endotoxin/LPS-induced prostaglandin synthesis by macrophages. We also measured production of nitric oxide (NO) that plays an important role in the cytotoxicity activity of macrophages towards microbial pathogens. After CF treatment of LPS-stimulated macrophages we found an up-regulation of TNF-α protein level in culture medium, no significant changes in the level of COX-2 protein expression and a decrease in NO production as well as in IL-1β and IL-6 release. Collectively, this series of experiments indicate that CF affect macrophage production of inflammatory mediators. However, further research is required in order to establish whether CF treatment can be beneficial in suppression of pro-inflammatory cytokine production and against progression of endotoxin-related diseases.

Keywords

Calcium fructoborateLipopolysaccharideInterleukins-1β and -6Tumor necrosis factor-αCyclooxygenase-2

Introduction

Boron has been implicated in a variety of metabolic and physiological processes in humans and animals [1]. Although accumulating evidence indicate a role for boron in the attenuation of inflammatory processes [24], the cellular mechanisms of its action have not been yet elucidated. The mediators released during the inflammatory process perpetuate the inflammatory response and are responsible of the clinical signs that are associated with inflammation. A great variety of mediators have been identified, including the products of the arachidonic acid (AA) metabolism, like prostaglandins, prostacyclin, and thromboxanes; the reactive oxygen intermediates (ROI); and cytokines such as TNF-α, and interleukins IL-1 and IL-6. The role that each of these mediators play in the inflammatory response varies depending on part of the stage of the inflammation during the mediator is released.

Discovery of naturally occurring boron complexes with organic compounds containing hydroxyl groups, sugars, adenosine-5-phosphate, pyridoxine, riboflavin, dehydroascorbic acid, and pyridine nucleotides led to the reassessment of the biochemical role of boron. The most stable esters are those in which boric acid is the bridge between two carbohydrate molecules, e.g., fructose–boron–fructose. Such soluble boron complexes are found naturally in phloem saps and plant nectars. Polysaccharides containing boron in similar linkages are found in plant cell walls in the form of pectins [5]. CF is most commonly found in fresh fruits and vegetables and is used under the commercial name FruiteX B as a nutritional supplement. It supports vitamin D metabolism, calcium metabolism, bone, and joint health and is involved in the regulation of steroid hormone levels and in healthy prostate maintenance [68]. We have previously investigated the effects of CF on human polymorphonuclear neutrophils (PMN) that play a central role in the inflammatory response [9]. Other previous in vitro studies from our laboratory suggested that this compound also displayed antioxidant and anti-tumor properties [10, 11].

In this paper, we examined the effects of CF on some inflammatory mediators’ production using LPS-stimulated murine macrophage RAW 264.7 cells as a cellular model. Thus, changes induced by CF in the dose range of 0.2–1 mM upon the release of cytokines such as TNF-α, IL-1β and IL-6, the level of COX-2 protein expression and upon production of NO have been evaluated.

Materials and Methods

CF was synthesized according to Miljkovic’s patent [6]. The purity of CF was checked by nuclear magnetic resonance and high-performance liquid chromatography, and the results indicated 99.9% analytical purity.

Cell Culture and Treatment

Murine macrophage RAW 264.7 cells, obtained from the American Type Culture Collection, were cultured at an initial density of 2.5 × 105 cells/cm2 into 24-well tissue culture plates in Dulbecco's Modified Eagle's Medium supplemented with 10% heat inactivated fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, and maintained in a humidified atmosphere with 5% CO2 at 37°C. They were activated for 24 h with 1 µg/ml LPS (Sigma-Aldrich Co.) in the absence or the continued presence of CF added to culture medium at concentrations of 0.2, 0.45, and 1 mM.

Cell Viability Assessment

Cell viability was assessed on cultured macrophages that were exposed to different treatments for the specified period of time. RAW 264.7 cells were incubated with 1 mg/ml MTT solution at 37ºC for 2 h. The yellow dye [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (MTT) is reduced to insoluble purple formazan granules by metabolically active cells. The precipitated formazan was dissolved in dimethyl sulfoxide, and after 10 min of slow shaking, the absorbance was recorded at 550 nm. Absorbance values, that are lower than those displayed by control cells, indicate a reduction in the cell activity and viability. Conversely, higher absorbance values indicate an increase in cell viability.

Cytotoxicity Assay

Murine RAW 264.7 macrophage cell line was routinely grown, treated, and assessed for leakage of lactate dehydrogenase (LDH) out of the cytoplasm of damaged cells. Briefly, after 24 h of various treatments with LPS ± CF, the culture media were recovered and used to measure the loss of cell viability and LDH release using an in vitro toxicology assay kit (TOX-7, Sigma-Aldrich Co.) according to the manufacturer’s instructions.

Nitrite Measurement

Nitrite levels in the culture media were determined using the Griess reaction. Briefly, 50 µl of cell culture medium was mixed with 100 µl of Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid, and 0.1% (w/v) naphthylenediamine-HCl] and incubated at room temperature for 10 min. The absorbance was then measured at 520 nm. Fresh culture media were used as blank for all experiments. Nitrite levels in samples were determined using a standard sodium nitrite curve.

ELISA-Based Cytokine Assay

The levels of TNF-α, IL-1β and IL-6 were determined using ELISA kits, purchased from Pierce Biotechnology Inc., following the manufacturer’s instructions. These assays were performed using the supernatants resulted by centrifugation of culture media collected at the end of cell culture.

Western Blotting for COX-2 Protein Expression

At the end of the culture period, cells were trypsinized, rinsed twice with PBS, and then lysed by sonication. Total cell lysates containing 25 μg protein, as determined by the Bradford method [12], were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide) and then transferred to nitrocellulose membranes. For COX-2 protein detection a WesternBreeze Chromogenic Western Blot Immunodetection kit (Invitrogen) was used. The mouse primary antibody against human COX-2 was purchased from Millipore.

Statistical Analysis

For the statistical evaluation of the obtained data, one-way ANOVA with Bonferroni’s multiple comparison tests was performed. All values are expressed as means ± SEM. Statistical analysis was performed using the Prism software (GraphPad, San Diego, CA).

Results

In a first step, cell viability after different treatments was studied using both MTT and LDH assays. As shown in Fig. 1, the treatment for 24 h with CF resulted in minor changes in cell viability of un-stimulated macrophages. Thus, the cell viability increased only by 4.2% and 5.2% in the case of macrophage treatment with 0.2 mM CF and 0.45 mM CF, respectively. On the contrary, the cell treatment with 1 mM CF decreased macrophage cell viability by 3.7%. LPS treatment induced about 11% reduction in cell viability. In addition, CF did not change the viability of LPS-activated macrophages. On the other hand, LDH assay into the culture media showed no significant differences between the un-stimulated and LPS-stimulated macrophages exposed to up to 1 mM CF (Fig. 2).
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Fig. 1

Effects of CF treatment in the dose range of 0.2–1 mM on cell viability of un-stimulated and stimulated (1 µg/ml LPS) RAW 264.7 macrophages. Data represent mean ± SEM of four experiments (each CF concentration was tested in triplicate). *p < 0.05 versus non-treated cells (NT), **p < 0.01 versus non-treated cells (NT)

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Fig. 2

Evaluation of cytotoxic effects of CF on RAW 264.7 macrophage cell line (un-stimulated and stimulated with 1 µg/ml LPS) by LDH assay. Data represent mean ± SEM of four experiments (each CF concentration was tested in triplicate)

In the study of possible anti-inflammatory effects of CF we considered the fact that upon activation, blood monocytes and tissue macrophages release a set of primary inflammatory mediators, such as IL-1β and TNF-α, thereby inducing the synthesis and secretion of several secondary cytokines such as IL-6. Recruitment of other immune effectors cells by chemotaxis then rapidly augments the local inflammatory response to counteract the inflammatory stimulus and to remove cellular debris associated with tissue damage [13].

Interleukin-1 is a primary mediator of immune responses to injury and infection, but the mechanism of its cellular release is unknown. IL-1 exists as two agonist forms (IL-1α and IL-1β) present in the cytosol of activated monocytes/macrophages. While IL-1α is predominantly membrane bound, IL-1β is secreted.

Our studies revealed a dose-dependent inhibition of mature IL-1β release with increasing doses of CF. The level of IL-1β induced by LPS decreased by 7% and 27%, after exposure to 0.45 mM and, respectively, 1 mM CF (Fig. 3).
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Fig. 3

Effects of treatments with increasing doses of CF on the release of mature IL-1β by LPS-stimulated RAW 264.7 macrophages. Data represent mean ± SEM of four experiments (each CF concentration was tested in triplicate). **p < 0.01 versus LPS-stimulated cytokine production, ***p < 0.001 versus LPS-stimulated cytokine production, filled diamond p < 0.001 versus spontaneous cytokine production

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in host defense due to its range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Although IL-6 is produced early in inflammation (shortly after IL-1 and TNF-α synthesis), this cytokine mediates both pro- and anti-inflammatory effects. Therefore, it is mandatory that molecular mechanisms modulating IL-6 action exist [14].

To get an insight into effects exerted by CF on the secretion of IL-6 by LPS-stimulated macrophages, an ELISA assay was used. Results of experiments presented in Fig. 4 indicate that addition of CF (0.2–1 mM) to cultured RAW 264.7 macrophages reduces secretion of IL-6 by LPS-stimulated cells, in a dose-dependent manner.
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Fig. 4

Effects of increasing doses of CF on the release of IL-6 from RAW 264.7 macrophages stimulated in vitro with 1 µg/ml LPS. Data represent mean ± SEM of four experiments (each CF concentration was tested in triplicate). ***p < 0.001 versus LPS-stimulated cytokine production, filled diamond p < 0.001 versus spontaneous cytokine production

Down-regulation of IL-6 protein level in culture media after CF treatment was more intensive by comparison with that registered for IL-1β (Fig. 3). Thus, the inhibition levels of IL-6 protein expression were about 45% at 0.2 mM CF, 81% at 0.45 mM CF, and 90% at 1 mM CF.

Another cytokine evaluated in this study was TNF-α, a protein produced in the early phase of inflammation in cells of reticuloendothelial origin such as macrophages. TNF-α mediates early-stage responses by regulating the production of other cytokines, including IL-1β and IL-6. Cumulative evidence indicates that abnormalities in the production or function of TNF-α play essential roles in many inflammatory lesions [15]. Our studies regarding the effects of CF treatments on TNF-α release by LPS-stimulated RAW 264.7 macrophages led to unexpected results. Thus, an enhancement of cytokine secretion in a dose-dependent manner was noticed (Fig. 5). Simultaneous treatment of RAW 264.7 macrophages with LPS and 1 mM CF induced an up-regulation of TNF-α level in the culture media with 279% in comparison with its level registered in the case of LPS-stimulated cells. In the absence of LPS stimulation, no statistical differences between the levels of TNF-α, IL-1β and IL-6 released by CF treated and untreated cells were noticed (data not shown).
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Fig. 5

Effects of CF on TNF-α production by LPS-stimulated RAW 264.7 macrophages. These cells were incubated with LPS (1 µg/ml) ± various doses of CF (0.2 mM; 0.45 mM; 1 mM) for 24 h. The level of this cytokine in culture media was quantified by an ELISA assay. ***p < 0.001 versus LPS-stimulated cytokine production, filled diamond p < 0.001 versus spontaneous cytokine production

Prostaglandin E2 and NO are two pleiotropic mediators produced at inflammatory sites by the inducible enzymes, COX-2 and nitric oxide synthase (iNOS), respectively. We investigated the effect of CF on the LPS-induced NO production by RAW 264.7 macrophages (Fig. 6). It can be observed that in un-stimulated cells a spontaneous production of NO occurs. The single treatment with LPS (1 μg/ml) induced a twofold increase in nitrite accumulation over basal level while macrophage associated treatment LPS/CF reduced the NO level in culture media by 10% (0.2 mM CF), 20% (0.45 mM CF), and 57.7% (1 mM CF).
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Fig. 6

Evaluation of CF effects on NO production by un-stimulated and stimulated (1 µg/ml LPS) RAW 264.7 macrophages. CF inhibits LPS-induced NO release from these cells. Data represent mean ± SEM of four experiments (each condition was tested in triplicate). ***p < 0.001 versus spontaneous NO production, filled diamond p < 0.001 versus LPS-stimulated NO production; three filled diamonds p < 0.001 versus LPS-stimulated NO production

Western blotting experiments revealed the constitutive protein expression of COX-2 in un-stimulated RAW 264.7 cells, the induction of COX-2 by LPS and the fact that the CF treatment did not affect LPS-induced COX-2 protein expression level (Fig. 7).
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Fig. 7

Western blotting detection of COX-2 protein showing that CF did not influence its expression level in LPS-stimulated RAW 264.7 cells. The experiment was performed in duplicate and similar results were obtained

Discussion and Conclusions

In the present study, we showed that treatment of LPS-stimulated RAW 264.7 macrophage cells with CF induced an inhibition of the IL-1β, IL-6 and NO release in the culture media, an increase of TNF-α production, and had no effects on LPS-induced COX-2 protein expression.

The pro-inflammatory cytokine IL-1β is synthesized by activated monocytes and macrophages as a 31-kDa, biologically inactive precursor that is proteolytically processed to the biologically active 17-kDa mature molecule by the IL-1β converting enzyme. Studies on LPS-stimulated cultured macrophages, showed that induction of apoptosis but not necrosis effectively induced conversion of the IL-1β precursor to its mature form and resulted in the concomitant release of the mature cytokine from the cell [16]. Our data suggest that CF affects the post-translational activation of biologically inactive IL-1β precursor. Considering the absence of pro-apoptotic effects of CF treatment (data not shown) we came to the conclusion that this compound could uncouple IL-1 β processing and apoptosis.

The effects of CF treatment on IL-6 synthesis by RAW 264.7 macrophages might be explained by the fact that IL-6 is a secondary cytokine whose expression can be stimulated by primary cytokines like IL-1, whose post-translational activation seems to be inhibited by this borate derivative. These studies provide evidence to support the view that CF can be an effective, safe anti-inflammatory agent.

Our results regarding CF effects on LPS-stimulated macrophage TNF-α production are contradictory because TNF-α plays a major role in regulating inflammation, mostly through the induction of inflammatory cytokines, including IL-1β and IL-6. Cao et al. [17] studied LPS-induced TNF-α formation in THP-1 cells and noticed the inhibitory effect exhibited by boric acid.

Interestingly, when Armstrong and Spears [3] examined the effect of boron supplementation of pig diets they found a decreased inflammatory response following a phytohemmaglutinin intradermal injection. They also noticed an increased level of TNF-α in serum as well as in peripheral blood monocytes isolated from pigs that received the B-supplemented diet and cultured in the presence of LPS. These data could not explain the reduction in localized inflammation following an antigen challenge in pigs. Other studies showed that boron increased TNF-α release by cultured human fibroblasts and chick embryo cartilage [18, 19]. The complex regulation of TNF-α synthesis, at the level of transcription, translation, and secretion, makes difficult to explain the high levels of this cytokine at the same time with the decrease in other inflammatory mediators [2022]. In addition, the signaling pathways involved in cytokine release from RAW 264.7 macrophages are now under investigation [23]. Moreover, the involvement of different pro- and anti-inflammatory mediators in a sequential and concerted manner and regulation of cytokine induction can occur after a variable pattern in different cell types and depends on the nature of the stimulatory ligand. These mediators can act at the level of cell surface, cell membrane, cytosol, or nucleus.

There is continuing interest in the effects of long-chain n – 3 polyunsaturated fatty acids (PUFAs) on human immune function and inflammatory processes [24, 25]. In previous studies [26, 27] it was shown that increasing dietary (n – 3) to (n – 6) fatty acid ratio from 0 to 1 resulted in a dose-response increase in TNF production by LPS-stimulated resident peritoneal macrophages. On the other hand, some experimental data suggested that boron had essential function similar to (n – 3) fatty acids [28]. Consequently, macrophage CF treatment might induce a replacement of n – 6 PUFA with n – 3 PUFA in cell membranes generating a decreased cellular response to inflammatory stimuli.

Nitric oxide is synthesized from l-arginine by l-arginine NO pathway and is converted to nitrite and nitrate in oxygenated solutions. A family of enzymes, termed the NO synthases (NOS), catalyze the formation of NO and citrulline from l-arginine, O2, and NADPH. The constitutive NOS isoforms (NOS-1 and NOS-3) produce low levels of NO as a consequence of increased intracellular Ca2+. By contrast, the inducible isoform of NOS (NOS-2 or iNOS) generates large amounts of NO through a Ca2+-independent pathway [29]. Some pro-inflammatory agents, such as endotoxin, TNF-α and IL-1 induce NOS-2 activity. High levels of NO induce changes suggestive of apoptosis in RAW 264.7 mouse macrophage cell line [30]. It is possible for CF to inhibit NO production by blocking iNOS expression in RAW 264.7 macrophages. Due to the critical role that NO release plays in mediating inflammatory responses, our data suggest that CF could represent a useful anti-inflammatory agent. Jeon et al. [31] showed that the p38 MAPK pathway is specifically involved in LPS-induced iNOS expression in LPS-stimulated RAW 264.7 cells. The p38 MAPK also regulates LPS-induced TNF-α and IL-1 production by monocytes [32, 33]. In a study focused on the effects of NO on TNF synthesis in the RAW 264.7 cell line [34] a suppression of LPS-induced TNF synthesis by exogenous addition of NO-releasing agents was found. The finding of an increased TNF production in the presence of two NO synthase inhibitors, indicated a negative feedback by endogenous NO on TNF synthesis in vitro.

Cyclooxygenase-2, the enzyme primarily responsible for induced prostaglandin synthesis, represents the product of an immediate early gene induced by endotoxin in macrophages. Secreted prostaglandins promote inflammation by increasing vascular permeability and vasodilation and by directing cellular migration into the site of inflammation through the production and release of pro-inflammatory cytokines such as interleukin-6 [35]. Our data suggest that CF does not affect COX-2 protein expression level in LPS-stimulated macrophages and, consequently, neither the prostaglandin synthesis, which might sustain the anti-inflammatory properties of this boron derivative.

Since CF complex is characterized by a high boron–fructose association constant (about 6,000) [36] we consider that in vitro response of LPS-stimulated macrophages is due to the entire molecule. There are studies demonstrating that various boron-containing compounds displayed anti-inflammatory properties [37, 38]. In a comparative study concerning the effects of CF and sodium borate on fMLP-stimulated PMN we found that CF exhibited superior anti-inflammatory and antioxidant properties [9]. Moreover, we demonstrated that while both boric acid and CF inhibited the growth of MDA-MB-231 breast tumor cells, only CF induced the apoptosis.

This study demonstrated that CF treatment of LPS-stimulated RAW 264.7 macrophages induced an up-regulation of TNF-α protein level in culture medium, no significant changes in the level of LPS-induced COX-2 protein expression and a decrease in NO production as well as in IL-1β and IL-6 release. Although we can conclude that CF might affect macrophage production of inflammatory mediators, these studies have to be completed with further research aiming to establish whether CF treatment can be beneficial for suppression of pro-inflammatory cytokine production and against progression of endotoxin-associated diseases. We also intend to elucidate the mechanism of CF effects and its efficacy as an anti-inflammatory agent comparing to drugs with well established anti-inflammatory action.

Copyright information

© Humana Press Inc. 2009