[Influence of nuclear factor-kappaB decoy oligonucleotides on RANTES expression and monocyte chemotactic activity in stromal cells of ectopic endometrium].
OBJECTIVE
To study the inhibitory effect on the expression of regulated upon activation, normal T cell expressed and secreted (RANTES) and monocyte chemotactic activity of ectopic endometrial stromal cells by nuclear factor (NF)-kappaB decoy oligonucleotides (ODN).


METHODS
The stromal cells of ectopic endometrium were divided into 3 groups. Two groups were cultured with or without 10 microg/L of interleukin (IL)-1beta. Another group was transfected with NF-kappaB decoy ODN with the aid of a lipofectamine reagent. After 4 h of transfection, 10 microg/L of IL-1beta was added to induce the stromal cells to secrete RANTES. Concentration of RANTES in the supernatant at 4, 8, 12, 24 and 36 h was measured with the sandwich enzyme linked immunosorbent assay (ELISA). U937 monocyte chemotactic activity was assayed in Boyden chambers. The specific RANTES-neutralizing monoclonal antibodies at serial doses (0.5, 1, 2, 4 and 8 mg/L) were added into IL-1beta induced medium of 24 h to detect the monocyte chemotactic activity of RANTES in supernatant.


RESULTS
The concentration of RANTES secreted by stromal cells was respectively (58 +/- 10), (150 +/- 35), (360 +/- 46) and (586 +/- 42) ng/L after IL-1beta stimulation for 8, 12, 24 and 36 h, significantly higher than that of stromal cells cultured without IL-1beta. The concentrations of RANTES were respectively (86 +/- 16), (128 +/- 28) and (183 +/- 32) ng/L after IL-1beta stimulation for 12, 24 and 36 h in stromal cells transfected with NF-kappaB decoy ODN, evidently lower than that of stromal cells stimulated with IL-1beta alone. The monocyte chemotactic index of 12, 24, 36 h in conditioned medium of stromal cells transfected with NF-kappaB decoy ODN was respectively 10.3 +/- 0.9, 13.7 +/- 1.1, 18.6 +/- 1.2, which was evidently lower than that of stromal cells stimulated with IL-1beta alone. The anti-RANTES antibody at 0.5, 1, 2, 4 and 8 mg/L inhibited respectively 5%, 23%, 40%, 62% and 61% of the chemotactic activity in 12 h medium treated with IL-1beta.


CONCLUSIONS
RANTES accounts for the majority of the monocyte chemotactic activity in IL-1beta induced medium of 24 h. NF-kappaB decoy ODN may influence the feed-forward inflammatory loop whereby IL-1beta from activated macrophages can lead to RANTES production by ectopic implants and further monocyte chemotaxis.
Wang
Xiu-li
XL
Department of Obstetrics and Gynecology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210036, China. xiuli_2266@163.com
Han
Su-ping
SP
Fang
Chun-li
CL
Mao
Yun-dong
YD
Wang
Wei
W
Lu
Jing
J
Zhang
Hong-mei
HM
D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000378:metabolism*; D002633:Chemotaxis; D004715:Endometriosis / Q000378:metabolism* / Q000473:pathology; D004717:Endometrium / Q000378:metabolism* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D009000:Monocytes / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics* / Q000378:metabolism; D017154:Stromal Cells / Q000378:metabolism / Q000473:pathology; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
C483024:CCL5 protein, human; D018946:Chemokine CCL5; D053583:Interleukin-1beta; D016328:NF-kappa B; C468119:NF-kappaB decoy; D009838:Oligodeoxyribonucleotides