Open Access, Peer-reviewed
pISSN 2288-6575
eISSN 2288-6796
    J Korean Surg Soc. 2011 Oct;81(4):229-234. English.
    Published online Oct 28, 2011.
    https://doi.org/10.4174/jkss.2011.81.4.229
    Copyright © 2011, the Korean Surgical Society
    Original Article

    Effect of hypertonic saline and macrophage migration inhibitory factor in restoration of T cell dysfunction

    Young-Hoon Yoon, Sung-Hyuk Choi, Yun-Sik Hong, Sung-Woo Lee, Sung-Woo Moon, Han-Jin Cho, Cheul Han,1 Young-Jin Cheon, ,1 and Vishal Bansal2
      • Department of Emergency Medicine, Korea University College of Medicine, Seoul, Korea.
      • 1Department of Emergency Medicine, Ewha Womans University School of Medicine, Seoul, Korea.
      • 2Division of Trauma and Surgical Critical Care and Burns, Department of Surgery, University of California San Diego School of Medicine, San Diego, CA, USA.
    • Correspondence to: Sung-Hyuk Choi. Department of Emergency Medicine, Korea University Guro Hospital, Korea University College of Medicine, 80 Guro 2-dong, Guro-gu, Seoul 152-703, Korea. Tel: +82-2-2626-1561, Fax: +82-2-2626-1562, Email: kuedchoi@korea.ac.kr
    Received April 13, 2011; Revised July 04, 2011; Accepted July 25, 2011.

    Journal of the Korean Surgical Society is an Open Access Journal. All articles are distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Purpose

    Trauma-induced suppression of cellular immune function likely contributes to sepsis, multiple organ dysfunction syndrome and death. T cell proliferation decreases after traumatic stress. The addition of prostaglandin E2 (PGE2), which depresses immune function after hemorrhage and trauma, to T-cells decreases T-cell proliferation; and hypertonic saline restores PGE2-induced T-cell suppression. Recently, it has become apparent that macrophage migration inhibitory factor (MIF) plays a central role in several immune responses, including T-cell proliferation. However, the role of MIF in mediating hypertonic saline (HTS) restoration of T cell dysfunction is unknown. Therefore, we hypothesize that T cell immune restoration by HTS occurs, at least in part, by a MIF-mediated mechanism.

    Methods

    Jurkat cells were cultured in Roswell Park Memorial Institute media, at a final concentration of 2.5 × 106 cell/mL. The effects of HTS on T-cell proliferation following PGE2-induced suppression were evaluated in Jurkat cells: HTS at 20 or 40 mmol/L above isotonicity was added. MIF levels were determined by enzyme-linked immunosorbent assay and western blot analysis.

    Results

    PGE2 caused a 15.0% inhibition of Jurkat cell proliferation, as compared to the control. MIF levels decreased in PGE2-suppressed cells, as compared to the control. MIF levels were higher in cells treated with HTS than PGE2-stimulated cells.

    Conclusion

    The role of HTS in restoring Jurkat cells proliferation suppressed by PGE2, at least in part, should be mediated through a MIF pathway.

    Keywords
    Hypertonic solutions; Macrophage Migration-Inhibitory factors; Prostaglandins E; Injuries; T-lymphocytes

    INTRODUCTION

    Immunological suppression is a well recognized consequence of trauma and hemorrhagic shock, and contributes to infectious complications, ultimately leading to sepsis and multiple organ dysfunction syndrome (MODS) [1]. There are several mechanisms of post-traumatic immune impairment, including T cell dysfunction. T cell dysfunction after traumatic stress is characterized by a decrease in T cell proliferation. The addition of prostaglandin E2 (PGE2), which depresses immune function after hemorrhage and trauma, to T cells decreases T-cell proliferation, while hypertonic saline (HTS) restores PGE2-induced T-cell suppression [2].

    Recently, it has become apparent that macrophage migration inhibitory factor (MIF) plays a central role in several immune responses, including the modulation of several cytokines and T-cell proliferation [3, 4]. By controlling immune and inflammatory responses, MIF is thought to play an important role in the pathophysiology of septic shock and chronic inflammatory diseases [5-7]. The role of MIF mediating HTS restoration of T cell dysfunction is unknown.

    Therefore, we hypothesize, that T cell immune restoration by HTS occurs, at least in part, by a MIF-mediated mechanism. The proposed experiments will provide the first evidence supporting MIF as the primary mechanism for restoring T cell dysfunction by HTS.

    METHODS

    Cells culture and cell stimulation

    Jurkat cells clone E6-1 (ATCC, Manassas, VA, USA) were maintained in Roswell Park Memorial Institute-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U/mL penicillin/streptomycin at 37℃ in a 5% carbon dioxide incubator. Cells were cultured to a density of 5 × 105 cell/mL. Cell viability, as determined by tyropan blue dye exclusion, was >99%. For the protein extracts, cells were plated at a density of 2.5 × 106 cell/mL in a 6-well flat-bottom culture plates, and were stimulated with PGE2 (1 µg/mL) (Sigma-Aldrich Co., St. Louis, MO, USA) in the presence or absence of HTS at 20 mmol/L (HTS20) and 40 mmol/L (HTS40) above isotonicity, resulting in sodium concentrations of 160 mmol/L and 180 mmol/L, which were measured by a GEM Premier 3000 (Instrumentation Laboratory, Lexington, MA, USA), respectively.

    Protein extracts

    After incubation for 24 hours at 37℃, the cells were washed 2 times in cold phosphate buffered saline (PBS) and then centrifuged for 10 minutes. Cells pellet were resuspended in 10 µL per 2 × 106 cell/mL of superlysis buffer (protease inhibitors, 1 M HEPES, 5 M NaCl, 0.5 M ethylenediaminetetraacetic acid, 1 mM NaOV4, 20% Triton X-100, 50 mM phenylmethylsulfonylfluoride), incubated on ice for 7 minutes, and then centrifuged at 3,000 × g for 15 minutes at 4℃. The supernatant was then transferred to an eppendorf tube and used for assay. The total protein concentration was determined by the Bradford method using a commercially available assay kit (Thermo Fisher Scientific, Rockford, IL, USA) [8]. The prepared protein lysates were aliquoted and used for Western blot analysis.

    T-cell proliferation assay

    Jurkat cells were plated in 96-well flat-bottom tissue culture plates to attain a final concentration of 2.5 × 106 cell/mL. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were conducted by the quantity of PGE2 0.01, 0.1, 1, 10 µg/mL and MTT assay value was the lowest at PGE2 of 1 µg/mL (have not shown). Therefere, PGE2 1 µg/ml were used. The effect of HTS on T-cell proliferation following PGE2-induced suppression was evaluated in Jurkat cells with 1 µg/mL PGE2, a concentration that suppresses T-cell proliferation, and HTS at 20 mmol/L and 40 mmol/L above isotonicity was added at the same time as PGE2. After incubation for 24 hours at 37℃, the resultant T-cell proliferation was determined by the MTT cell proliferation assay (ATCC).

    Enzyme-linked immunosorbent assay (ELISA) for MIF

    MIF concentration in culture supernatants was measured by sandwich enzyme-linked immunosorbent assay. Briefly, 2 µg/mL of monoclonal capture antibody (R&D Systems, Minneapolis, MN, USA) was added to a 96-well plate and incubated for 2 hours at room temperature. After incubation, the plates were incubated in blocking solution comprised of PBS containing 1% bovine serum albumin (BSA) and 0.05% Tween 20 for 2 hours at room temperature. The test samples and standard recombinant MIF (R&D Systems) were added to the plates, and the plates were incubated for overnight at 4℃. The plates were then washed four times with PBS containing Tween 20, 200 ng/mL of biotinylated detection monoclonal antibodies (R&D Systems) was added, and the plates were incubated for 2 hours at room temperature. Subsequently, the plates were washed, streptavidin-alkaline-phosphatase (1:2,000; Sigma-Aldrich Co.) was added, and the reaction was allowed to proceed for 2 hours at room temperature. The plates were washed four times, and 1 mg/mL of p-nitrophenylphosphate dissolved in diethanolamine (Sigma-Aldrich Co.) was added to induce the color reaction, which was stopped by adding 50 µL of 1 N NaOH. The optical density at 405 nm was measured on an automated microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA). A standard curve was generated by plotting optical density versus the log of the MIF concentration. The experiments were conducted 10 times.

    Western blot analysis for MIF expression

    Expression of MIF protein was quantified by Western blot analysis. Proteins (20 µg/sample) were fractionated on an 15% sodium dodecylsulfate-polyacrylamide gel (Bio-Rad Laboratories Inc.) and transferred onto a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% BSA (Sigma-Aldrich Co.), and then incubated with a primary antibody, anti-human MIF (1:250; R&D systems). After washing, membranes were incubated with 1:2000 horseradish peroxidase-labeled goat anti-mouse antibody (R&D systems) as the secondary antibody. The proteins were detected using SuperSignal (Thermo Fisher Scientific Inc., Rockford, IL, USA) chemiluminescence kit.

    Statistical analysis

    One-way analysis of variance was performed to evaluate the significance of difference between the experimental groups. For a single comparison of two groups, a Student's t-test was used with SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as mean ± SD. Values of P < 0.05 were considered significant. All experiments were performed in triplicate.

    RESULTS

    The effect of HTS on PGE2-induced Jurkat cell suppression

    Jurkat cells were plated on 96-well culture plates at a concentration of 2.5 × 106 cell/mL in cell culture media. PGE2 at 1 µg/mL inhibited Jurkat cell proliferation by 14.7% (P < 0.05) (Fig. 1). The addition of HTS restored Jurkat cell proliferation suppressed by PGE2 to control levels, as measured using an MTT cell proliferation assay (P < 0.05). There was no statistical difference in restoration by either HTS20 or HTS40 (Fig. 1).

    Fig. 1
    Prostaglandin E2 (PGE2) inhibited Jurkat cell proliferation by 14.7% (P < 0.05). The addition of HTS restored Jurkat cell proliferation suppressed by PGE2 to control levels, as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) cell proliferation assay (P < 0.05). There was no statistical difference in restoration by either HTS20 or HTS40. The experiments were conducted 10 times and conducted by MTT. The experiments were also done by EZ-Cytox Cell viability assay kit (Daeil Lab, Seoul, Korea) and the results were the similar. ANOVA, analysis of variance. a)Mean ± SD (ANOVA, Paired t-test) P < 0.05, 2.5 × 106 cell/mL 1 day incubation number: 10 times.

    The effect of HTS on MIF concentrations in the cell supernatant

    To determine the relation between HTS and MIF on Jurkat cell proliferation, MIF levels were measured in the cell supernatant. The MIF level was decreased by 0.98 ng/mL ± 0.38 in the supernatant of PGE2 stimulated cells, when compared to control levels (1.19 ng/mL ± 0.48). On the other hand, HTS20 restored and increased the MIF level (1.09 ng/mL ± 0.38) in PGE2-suppressed Jurkat cells (P < 0.05). PGE2-suppressed Jurkat cells treated with HTS40 had a higher MIF level (1.16 ng/mL ± 0.33) than HTS20-treated cells, however, there was not a statistically significant difference of MIF levels in the supernatant of HTS20 or HTS40-treated cells (Fig. 2).

    Fig. 2
    Migration inhibitory factor (MIF) levels in cell supernatants were measured. Jurkat cells in presence of prostaglandin E2 (PGE2) had a decrease in MIF level (0.98 ng/mL ± 0.38), when compared with Jurkat cells alone (1.19 ng/mL ± 0.48). HTS20 restored and increased MIF level (1.09 ng/mL ± 0.38) when compared with PGE2-suppressed Jurkat cells (P < 0.05). PGE2-suppressed Jurkat cells treated with HTS40 had the highest MIF level (1.16 ng/mL ± 0.33): however, there was no statistically significant difference in MIF levels between the HTS20- and HTS40-treated groups. The experiments were conducted 10 times. ANOVA, analysis of variance. a)Mean ± SD (ANOVA, Paired t-test) P < 0.05, 2.5 × 106 cell/mL 1 day incubation number: 10 times.

    The effect of HTS on MIF expression

    To determine the HTS effect on MIF expression, western blot analysis was performed. Correlating with the ELISA, levels of MIF protein expression were lower in PGE2-stimulating cells (16% decrease in band density) (P < 0.05). The addition of HTS to PGE2-stimulated cells increased MIF protein expression, with the highest expression in the HTS40-treated cells, however, there was not a statistically significant difference of MIF protein expression in the HTS20 or HTS40-treated cells (Fig. 3).

    Fig. 3
    Levels of migration inhibitory factor (MIF) protein expression were lower in prostaglandin E2 (PGE2)-stimulated cells (16% decrease in band density) (P < 0.05). The addition of hypertonic saline (HTS) to PGE2-stimulated cells increased MIF protein expression with the highest expression in the HTS40-treated cells. ANOVA, analysis of variance. a)Mean ± SD (ANOVA, Paired t-test) P < 0.05, 2.5 × 106 cell/mL 1 day incubation MIF MW: 12.5 KDa number: 5 times.

    DISCUSSION

    Trauma-induced suppression of cellular immune function likely contributes to sepsis, MODS, and death. This suppression is secondary to several immunomodulating factors including PGE2, transforming growth factor beta (TGFβ), interleukin 4 (IL-4), and IL-10, which block certain intracellular signaling events in T-cells [1]. T-cell dysfunction after traumatic stress is characterized by a decrease in T-cell proliferation, an aberrant cytokine profile, decreased T-cell monocyte interactions, and attenuated expression of the T-cell Receptor Complex [9-11]. The exact mechanism involved in the development of T-cell dysfunction is unknown. Under stressful conditions, macrophage are triggered to rapidly produce and release PGE2, a powerful endogenous immune suppressant [12]. PGE2 interacts with the corresponding membrane receptor of T cells, blocking T cell function by interfering with IL-2 gene expression at multiple stages [13-15]. In addition, PGE2 is a potent activator of cAMP-dependent protein kinase, which regulates expression of p27kip1 and cyclin D3 to suppress proliferation of leukemic T cell lines, including Jurkat cells, by arresting cells in the G1 phase of the cell cycle [15]. Therefore, the experiments were performed with PGE2, as it has been shown that PGE2, at a concentration of 1 µg/mL, causes inhibition of T cell proliferation by about 15%. Moreover, HTS restored suppressed PGE2-induced T cell proliferation. Hyperosmolality may play an important role in the HTS-enhancement of T-cell proliferation and hypertonicity increased IL-2 expression in T-cell proliferation [16-18]. However, the mechanism explaining the HTS-mediated restoration of T cell function is still unclear.

    MIF may serve as a general marker for systemic inflammation in septic and non-septic acute critical illness [5-7]. Furthermore, MIF is a cytokine that is secreted by the anterior pituitary and immune cells in response to surgical stress, injury, and sepsis. This cytokine appears to be a critical regulator of the inflammatory pathways, leading to systemic inflammatory response syndrome and subsequent MOSF [4, 19]. Besides, activated T cells produce MIF, and neutralizing anti-MIF antibodies inhibit T-cell proliferation and IL-2 production. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation, as well as interleukin 2 and interferon γ production [3]. And MIF also reduces pro-oxidative stress-induced apoptosis and nitric oxide induced apoptosis. As to a contribution by MIF to the regulation of cell survival, the inhibitory effect of MIF on apoptosis could correlate with its reported stimulatory effects on cell proliferation, even though cellular apoptosis and cell proliferation do not simply oppose each other [20-22].

    That is to say, MIF exhibits pro- and anti-inflammatory activities, and regulates cell proliferation and survival. Therefore, we know that T cell immune restoration by HTS occurs, at least in part, by a MIF-mediated mechanism. In our experiment, PGE2 inhibited Jurkat cell proliferation and decreased MIF levels, when compared to control levels. The addition of HTS increased MIF production, as compared with PGE2-stimulated Jurkat cells in concordance with restored PGE2-suppressed Jurkat cell proliferation. Considering side effect of HTS, HTS at sodium concentrations of 160 mM showed sufficient result in this experiment. It has been suggested that HTS could be a proliferation regulator by a MIF-mediated mechanism. In order words, PGE2 reduces expression of cyclin D and induces expression of p27kip1, while MIF regulates the expression of the cell cycle regulators cyclin and p27 [15, 23, 24]. Therefore, HTS could restore PGE2-suppressed Jurkat cell proliferation through MIF production. Cho et al. [25] showed that NF-kB inhibition leads to increased synthesis and secretion of MIF in human CD4+ T cells. Notably, HTS also inhibits NF-κB, as compared to isotonic saline, in our experiment (unpublished results). However, the mechanism of MIF regulation of HTS is not yet explained.

    Unfortunately, the present study is associated with certain limitation. First, PGE2 were used alone as immunosuppressant in present study. If another immuno-suppressant (IL-4, IL-10, et al.) were used, the present study might have more interesting outcome. Second, This study was done about MIF only, so other cytokine were not used. And ELISA and western blot analysis were performed for MIF levels. However, if another cytokine and another method for MIF levels (realtime PCR on the expression of mRNA for MIF) are used, the present study might have more interesting outcome, therefore, we will consider conducting further experiments by using other cytokine and methods in the future.

    The role of HTS in restoring Jurkat cell proliferation suppressed by PGE2, at least in part, should be mediated through a MIF pathway. Further studies are needed to determine the effects of HTS and MIF.

    Notes

    No potential conflict of interest relevant to this article was reported.

    ACKNOWLEDGEMENTS

    This work was partially supported by a Korea University Grant and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (R1009981).

    References

      1. Napolitano LM, Faist E, Wichmann MW, Coimbra R. Immune dysfunction in trauma. Surg Clin North Am 1999;79:1385–1416.
      1. Coimbra R, Junger WG, Liu FC, Loomis WH, Hoyt DB. Hypertonic/hyperoncotic fluids reverse prostaglandin E2(PGE2)-induced T-cell suppression. Shock 1995;4:45–49.
      1. Bacher M, Metz CN, Calandra T, Mayer K, Chesney J, Lohoff M, et al. An essential regulatory role for macrophage migration inhibitory factor in T-cell activation. Proc Natl Acad Sci U S A 1996;93:7849–7854.
      1. Bernhagen J, Calandra T, Bucala R. Regulation of the immune response by macrophage migration inhibitory factor: biological and structural features. J Mol Med (Berl) 1998;76:151–161.
      1. Lee JY, Song SU, Shin SH. Normal serum macrophage migration inhibitory factor concentrations of Korean people according to age. J Korean Geriatr Soc 2004;8:165–169.
      1. Lehmann LE, Novender U, Schroeder S, Pietsch T, von Spiegel T, Putensen C, et al. Plasma levels of macrophage migration inhibitory factor are elevated in patients with severe sepsis. Intensive Care Med 2001;27:1412–1415.
      1. Lehmann LE, Weber SU, Fuchs D, Klaschik S, Schewe JC, Book M, et al. Intracellular detection of macrophage migration inhibitory factor in peripheral blood leukocytes. Free Radic Biol Med 2005;38:1170–1179.
      1. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.
      1. Smith JW, Gamelli RL, Jones SB, Shankar R. Immunologic responses to critical injury and sepsis. J Intensive Care Med 2006;21:160–172.
      1. Lee JS, Han C, Cho SN, Chung SY. Effect of rapamycin on the cell cycle arrest of T lymphocytes. J Korean Surg Soc 2007;73:13–20.
      1. Park MH, Lee CS, Cho MH, Han C, Koh YS, Kim JC, et al. In vitro induction of carcinoembryonic antigen (CEA) specific cytotoxic T lymphocytes using dendritic cells pulsed with CEA peptide. J Korean Surg Soc 2005;69:359–366.
      1. Santoli D, Phillips PD, Colt TL, Zurier RB. Suppression of interleukin 2-dependent human T cell growth in vitro by prostaglandin E (PGE) and their precursor fatty acids. Evidence for a PGE-independent mechanism of inhibition by the fatty acids. J Clin Invest 1990;85:424–432.
      1. Roper RL, Phipps RP. Prostaglandin E2 regulation of the immune response. Adv Prostaglandin Thromboxane Leukot Res 1994;22:101–111.
      1. Anastassiou ED, Paliogianni F, Balow JP, Yamada H, Boumpas DT. Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2R alpha gene expression at multiple levels. J Immunol 1992;148:2845–2852.
      1. van Oirschot BA, Stahl M, Lens SM, Medema RH. Protein kinase A regulates expression of p27(kip1) and cyclin D3 to suppress proliferation of leukemic T cell lines. J Biol Chem 2001;276:33854–33860.
      1. Hoyt DB, Junger WG, Loomis WH, Liu FC. Effects of trauma on immune cell function: impairment of intracellular calcium signaling. Shock 1994;2:23–28.
      1. Junger WG, Liu FC, Loomis WH, Hoyt DB. Hypertonic saline enhances cellular immune function. Circ Shock 1994;42:190–196.
      1. Choi SH, Bansal V, Costantini T, Putnam J, Loomis W, Coimbra R. Arginine is essential in reversing prostaglandin E(2) T-cell suppression by hypertonic saline. J Surg Res 2009;156:83–89.
      1. Larson DF, Horak K. Macrophage migration inhibitory factor: controller of systemic inflammation. Crit Care 2006;10:138.
      1. Lue H, Kleemann R, Calandra T, Roger T, Bernhagen J. Macrophage migration inhibitory factor (MIF): mechanisms of action and role in disease. Microbes Infect 2002;4:449–460.
      1. Mitchell RA, Liao H, Chesney J, Fingerle-Rowson G, Baugh J, David J, et al. Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: regulatory role in the innate immune response. Proc Natl Acad Sci U S A 2002;99:345–350.
      1. Nguyen MT, Lue H, Kleemann R, Thiele M, Tolle G, Finkelmeier D, et al. The cytokine macrophage migration inhibitory factor reduces pro-oxidative stress-induced apoptosis. J Immunol 2003;170:3337–3347.
      1. Kleemann R, Hausser A, Geiger G, Mischke R, Burger-Kentischer A, Flieger O, et al. Intracellular action of the cytokine MIF to modulate AP-1 activity and the cell cycle through Jab1. Nature 2000;408:211–216.
      1. Mitchell RA, Bucala R. Tumor growth-promoting properties of macrophage migration inhibitory factor (MIF). Semin Cancer Biol 2000;10:359–366.
      1. Cho ML, Moon YM, Heo YJ, Woo YJ, Ju JH, Park KS, et al. NF-kappaB inhibition leads to increased synthesis and secretion of MIF in human CD4+ T cells. Immunol Lett 2009;123:21–30.
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    MeSH Terms
    Blotting, Western
    Cell Proliferation
    Dinoprostone
    Enzyme-Linked Immunosorbent Assay
    Hemorrhage
    Humans
    Hypertonic Solutions
    Jurkat Cells
    Macrophage Migration-Inhibitory Factors
    Macrophages
    Multiple Organ Failure
    Negotiating
    Prostaglandins E
    Sepsis
    T-Lymphocytes
    Metrics
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