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Ahn, Cha, Koo, Bae, Yoo, Song, Ahn, Hwang, Park, and Yang: In vitro suppression of natural killer cell cytolytic activities by prednisolone in patients with history of unexplained recurrent spontaneous abortion and elevated peripheral blood NK cell fraction
Original Article
Korean Journal of Obstetrics & Gynecology

Korean Journal of Obstetrics & Gynecology 2011; 54(7): 361-368.

Published online: 18 July 2011

DOI: https://doi.org/10.5468/KJOG.2011.54.7.361

In vitro suppression of natural killer cell cytolytic activities by prednisolone in patients with history of unexplained recurrent spontaneous abortion and elevated peripheral blood NK cell fraction

Hyun Suk Ahn, MD1, Sun Hwa Cha, MD1, Hwa Seon Koo, MD1, Ju Youn Bae, MD1, Ji Hee Yoo, MD1, In Ok Song, MD1, Hyun Kyoung Ahn, MD1, Su Jin Hwang, MS2, Dong-Wook Park, PhD3, Kwang Moon Yang, MD1

1Department of Obstetrics and Gynecology, Cheil General Hospital, Women's Healthcare Center, Kwandong University College of Medicine, Seoul, Korea.

2Laboratory of Molecular Oncology, Cheil General Hospital, Women's Healthcare Center, Kwandong University College of Medicine, Seoul, Korea.

3Laboratory of Reproductive Biology and Infertility, Cheil General Hospital, Women's Healthcare Center, Kwandong University College of Medicine, Seoul, Korea.

Corresponding author: Kwang Moon Yang, MD, PhD. Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Health Care Center, Kwandong University College of Medicine, 1-19 Mukjeong-dong, Jung-gu, Seoul 100-380, Korea. Tel: +82-2-2000-7545, Fax: +82-2-2000-7790, km1yang@naver.com

Received 7 March 2011       Revised 17 April 2011       Accepted 10 June 2011

Copyright © 2011. Korean Society of Obstetrics and Gynecology

(open-access):

This is an Open Access article 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

Objective

We aimed to evaluate the effect of prednisolone (PDS) on natural killer cell (NK cell) cytolytic activity in vitro.

Methods

Blood samples from 74 patients with history of unexplained recurrent spontaneous abortion who elevated peripheral blood NK cell fraction were collected prospectively. Peripheral blood monocytes which containing NK cells were isolated and separated to three different tubes which containing target (K562) cells by the 50:1 effector to target (E:T) ratio. PDS or intravenous immunoglobulin (IVIG) was additionally added to 2 tubes for evaluate their suppressive effect. The percentage killing of target cells was recorded numerically by using flow cytometer and the values between groups were statistically analyzed.

Results

The mean target cell killing percentage was 40.5% in co-culture tube which was not added PDS or IVIG. In culture experiments which was added IVIG, the killing percentage is reduced to 37.7% which showed no significant differences compared to that of co-cultured tube which was not added PDS or IVIG. But, in experiments with added PDS, the killing percent was reduced to 19.5% and the difference was statistically significant (P<0.001) compared to that of co-cultured tube which was not added PDS or IVIG. On comparing the reduction in killing percentage of target cells by PDS and IVIG, statistically significant reduction in the PDS coculture was noted (P<0.005).

Conclusion

NK cell cytolytic activity is effectively down-regulated by using PDS in vitro. Moreover, the effect of PDS in down-regulation of NK cell cytolytic activity is seems to superior than that of IVIG. But, large scaled in vivo study is needed.

Keywords: Recurrent miscarriage, Natural killer cell cytolytic activity, Prednisolone

Figures and Tables

Fig. 1
Determination of immune cell profiles in peripheral blood. Gating strategy for three color flow cytometry evaluation of peripheral blood immune cell. This step descript of analysis gate around lymphocyte (R1), excluding cell debris (A). Next step shows four regions as NK cell; CD3-/16+/56+. (B) NKT cell; CD3+/16+/56+, T cell; CD3+/56- and B cell (CD3-/56-) each. SSC, side scatter; PI, propidum iodie dye; PKH2, green fluorescent cell linker dye; NKT, natural killer T cell; NK, natural killer.
kjog-54-361-g001
Fig. 2
Determination of peripheral blood natural killer cell cytolytic activities. Figures describes analysis gate around K562 (target cell) live & dead cell in control (A, no effector cell), 50:1 effector cell:target cell (E:T) ratio (B), 25:1 E:T ratio (C), and 12.5:1 ET ratio (D) each which excluding cell debris. PKH2, green fluorescent cell linker dye; PI, propidum iodie dye.
kjog-54-361-g002
Fig. 3
Mean percentage of target cell killed with different effector to target cell ratios. aP<0.05, analysis of variance.
kjog-54-361-g003
Fig. 4
Mean percent to target cells with intravenous immunoglobulin or prednisolone coculture. IVIG, intravenous immunoglobulin; PDS, prednisolone. aP<0.05, analysis of variance (ANNOVA). bP<0.05, ANNOVA.
kjog-54-361-g004
Table 1
Clinical characteristics and peripheral blood immune cell profiles in study population
kjog-54-361-i001

NKT, natural killer T cell; NK, natural killer.

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