J Korean Surg Soc.  2012 Jan;82(1):1-7. 10.4174/jkss.2012.82.1.1.

Hypertonic saline downregulate the production level of lipopolysaccharide-induced migration inhibitory factor in THP-1 cells

• Han C
• Choi SH
• Yoon YH
• Cho YD
• Kim JY
• Hong YS
• Lee SW
• Moon SW
• Cho HJ
• Cheon YJ

• KMID: 1437748
• DOI: http://doi.org/10.4174/jkss.2012.82.1.1

Abstract

PURPOSE
Macrophage migration inhibitory factor (MIF) may serve as a general marker for systemic inflammation in septic and nonseptic acute critical illness. Additionally, our previous experiment has demonstrated that immunosuppressant Prostaglandin E2 (PGE2) lowered MIF levels and inhibited T-cells proliferation when compared to control levels. The addition of hypertonic saline (HTS) increased MIF production as compared with PGE2-stimulated T-cells in concordance with restore PGE2-suppressed T-cells proliferation. Generally, HTS has been well known for its anti-inflammatory effect so far. Therefore, the experiments were conducted to evaluate MIF after stimulating lipopolysaccharide (LPS) either in the presence or absence of HTS in monocyte, in response to early phase injury.
METHODS
Human acute monocytic leukemic cell line (THP-1) cells were cultured in RPMI media, to a final concentration of 1 x 10(6) cells/mL. The effect of HTS on LPS-induced MIF was evaluated in monocyte with 1 microg/mL LPS. HTS at 10, 20 or 40 mmol/L above isotonicity was added. MIF concentrations of the supernatant were determined by enzyme-linked immunosorbent assay, and cell lysates were used for Western blots analysis to determine the MIF expression.
RESULTS
MIF concentrations in the cell supernatant increased in LPS-induced cells compared to control cells. Also, levels of MIF protein expression were higher in LPS stimulating cells. However, the addition of HTS to LPS stimulated cell restored MIF concentrations and MIF expression.
CONCLUSION
The role of HTS in maintaining physiological balance in human beings, at least in part, should be mediated through the MIF pathway.

Keyword

Hypertonic saline solution; Macrophage migration-inhibitory factors; Lipopolysaccharides; Anti-inflammatory agents; Immunosuppression

MeSH Terms

Anti-Inflammatory Agents
Blotting, Western
Cell Line
Critical Illness
Dinoprostone
Enzyme-Linked Immunosorbent Assay
Humans
Immunosuppression
Inflammation
Lipopolysaccharides
Macrophage Migration-Inhibitory Factors
Macrophages
Monocytes
Saline Solution, Hypertonic
T-Lymphocytes
Anti-Inflammatory Agents
Dinoprostone
Lipopolysaccharides
Macrophage Migration-Inhibitory Factors
Saline Solution, Hypertonic

Figure

• 

  Fig. 1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay in THP-1 cells after 24 hours treatment with lipopolysaccharide (LPS) and varying concentrations of hypertonic saline (HTS). Absorbance of MTT formazan was determined at 570 nm using enzyme-linked immunosorbent assay reader. *LPS induced THP-1 cell viability by 19.3% (P < 0.05). However, HTS alone has no cytotoxic effects in THP-1 cells. Besides, †HTS partially protects against LPS-induced cytotoxicity in THP-1 cells. There was no statistical difference in protective effect by either HTS20 or HTS40.

• 

  Fig. 2 Migration inhibitory factor (MIF) levels in cell supernatant were measured. THP-1 cells in presence of lipopolysaccharide (LPS) had increase in MIF level (0.69 ng/mL ± 0.05) when compared with THP-1 cells (0.05 ng/mL ± 0.01) at 2 hours. However, hypertonic saline (HTS) 10 restored partially and decreased MIF levels (0.49 ng/mL ± 0.05) when compared with LPS-stimulated THP-1 cells (P < 0.05). LPS-stimulated THP-1 cells treated with HTS40 had lower MIF levels (0.27 ng/mL ± 0.04) than HTS10-treated cells, however, there was no statistically significant difference on MIF levels according to HTS concentrations or measured times. ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 3 Levels of Migration inhibitory factor (MIF) protein expression were higher in lipopolysaccharide (LPS)-stimulating cells (25% decrease in band density) (P < 0.05). Addition of hypertonic saline (HTS) to LPS-stimulated cell decreased MIF protein expression regardless of HTS concentrations.

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