Molecular Human Reproduction, Vol. 7, No. 8, 787-790,
August 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Nuclear factor-kappa B is essential for up-regulation of interleukin-8 expression in human amnion and cervical epithelial cells
1 Institute of Obstetrics and Gynaecology, Imperial College School of Medicine, Queen Charlotte's Hospital, Goldhawk Road, London W6 0XG, UK and 2 Division of Gastroenterology, Department of Internal Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6144, USA.
Abstract
Interleukin-8 (IL-8) is a cytokine which recruits and activates neutrophils into tissue stroma. It is present in uterine tissues and its concentration increases in the third trimester and with labour. The promoter region of the
IL-8
gene contains binding sites for the transcription factors, nuclear factor-kappa B (NF-
B), activator protein-1 (AP-1) and CCAAT/enhancer-binding protein (C/EBP). These are in close proximity to each other and to the coding region of the gene. This study used site-directed mutagenesis of each of these sites to examine the relative importance of each site in
IL-8
gene expression in a cervical cell line and in amnion cells obtained before and after labour. We found that the NF-B site was essential for basal and IL-1ß-stimulated gene expression in all cell types. Neither of the other binding sites was consistently essential for gene expression but may have an additive role in promoter activity. We conclude that the NF-B binding site is essential for up-regulation of
IL-8
gene expression in these uterine cell types. An increase in IL-8 expression has been shown to occur in the uterus in association with parturition and NF-B binding to the promoter may be of importance at this time.
amnion/cervix/interleukin-8/NF-kB/parturition
Introduction
Interleukin-8 (IL-8) is a chemokine of the CXC type that is a potent attractor and activator of neutrophils (Baggiolini
et al
., 1989
). It is present within the uterus in cervix (
Barclay et al., 1993
), placenta (
Elliott et al., 1998
), myometrium (
Elliott et al., 2000
), amnion and choriodecidua (
Kelly et al., 1992
). Its production by fetal membranes, placenta, myometrium and lower segment fibroblasts has been shown to increase in the late third trimester or with labour at term (
Winkler et al., 1998
,
1999
). We have shown that IL-8 in amnion is increased in the third trimester and after labour at term and that its expression in the choriodecidua shows parallel increases (unpublished data). An influx of neutrophils has been shown to occur into the uterine cervix and myometrium (
Junqueira et al., 1980
; Thomson, 1999) in association with labour and it is thought that IL-8 plays a key role in this neutrophil recruitment. The cervical changes of effacement and dilatation that occur with labour are due to the release of metalloproteinases, such as MMP-8 (neutrophil elastase), from activated neutrophils (
Osmers et al., 1992
). IL-8 thus plays a key role in the events of parturition.
The promoter region of the IL-8 gene contains functional binding sites for the transcription factors (NF-B), activator protein (AP-1) and CCAAT/enhancer-binding protein (C/EBP) in close proximity within 130 bp of the coding region (Figure 1
). Located upsteam of these sites are two glucocorticoid response elements (GRE) (
Mukaida et al., 1989
). In other cell types it has been shown that the NF-B site is essential for
transcription (Stein et al., 1993;
Kunsch et al., 1994
;
Wu et al., 1997
). The role for the AP-1 and C/EBP sites varies between the tissues studied.
|
The aim of this study was to determine the role of the transcription binding sites for NF-
B, AP-1 and C/EBP in regulation of basal and stimulated
IL-8
gene expression in primary amnion cells obtained before and after labour at term and in the human cervical epithelial cell line HOG-1 (
Gleeson et al., 1990
). Materials and methods
IL-8–luciferase reporter constructs
The constructs used were as previously described (Wu, 1997). In brief, the promoter region of the
IL-8
gene (–135/+46bp) was amplified by polymerase chain reaction and the fragment ligated into the luciferase reporter plasmid pGL2-Basic (Promega, Southampton, UK) to give the wild type construct (wt)LUC. Three further constructs were developed with site-directed mutations at the AP-1, NF-B and C/EBP binding sites to produce (mAP-1)LUC, (mNF-B)LUC and (mC/EBP)LUC respectively (Figure 1
).
Amnion cell cultures and transient transfections
Amnion was obtained from elective Caesarean section (L–) before labour at term or after spontaneous vaginal delivery at term (L+). Ethical approval for the collection of the tissues was obtained from the Hammersmith Hospital LREC. Term was defined as 37–42 completed weeks of pregnancy. There was no clinical evidence of infection or meconium staining in any of the samples obtained. Elective Caesarean section was performed for maternal indications, such as breech presentation or previous Caesarean or uterine surgery or maternal request. There was no uterine activity prior to surgery. Amnion was digested and cell cultures were established as previously described (Bennett, 1987). Cells were grown to 70–80% confluence and maintained serum-free prior to transfection. Transfection conditions were optimized for charge ratio in each cell type. 1 µg plasmid DNA was transfected into each well using a liposome-mediated method with Tfx-50 reagent (Promega, Southampton, UK). Cells were cultured overnight and then either left unstimulated or stimulated with IL-1ß (1 ng/ml) for 6 h. Cytoplasmic extract of the cells was prepared and analysed for luciferase activity with a commercially available kit (Promega, Southampton, UK).
HOG-1 cell culture and transient transfections
Human cervical epithelial cells (HOG-1) (
Gleeson et al., 1990
) were cultured to 70–80% confluence. Charcoal-stripped serum was added to the medium used prior to transfection. Transient transfection with each construct was performed as described above except that 0.5 µg plasmid DNA was used in each well.
Reporting of results and statistics
Three replicates were studied for each cell type and three samples of each were used. Results are expressed as percentage of the unstimulated wild-type promoter activity. Significance was determined using an ANOVA test with Fisher's protected least significant difference (PLSD) post-hoc analysis and significance was determined at the 95% level.
Results
Transient transfection of a promoterless `empty vector' generated negligible luciferase activity. Transient transfection of the wild type construct resulted in luciferase activity in each of the cell types studied. This was significantly increased by stimulation with IL-1ß.
HOG-1 cells
In these immortalized cervical epithelial cells, mutation of the NF-B site significantly reduced the unstimulated reporter expression (
P
< 0.0001
) and prevented the increase in expression by IL-1ß. Mutation of the C/EBP and the AP-1 sites also significantly repressed constitutive promoter activity (
P
< 0.0001
). Mutation of the AP-1 site did not prevent significant stimulation of promoter expression by IL-1ß. Cells transfected with the C/EBP mutated construct showed a slight increase in reporter expression after IL-1ß stimulation but the difference was not significant (Figure 2
).
|
Pre-labour amnion cells
Primary cell cultures transiently transfected with the mutated NF-
B construct also demonstrated reduced basal reporter activity as compared with those transfected with the wild type construct (
P
< 0.005
). This mutation prevented further stimulation in promoter activity by IL-1ß. In these cells mutation of the C/EBP or the AP-1 sites did not alter unstimulated activity, while small increases in promoter activity (significant for the AP-1 mutation but not for the C/EBP mutation) occurred after IL-1ß stimulation (Figure 3
).
|
Post-labour amnion cells
Transient transfection of these primary cells with any of the three mutated constructs did not significantly alter the unstimulated reporter expression. Increased reporter expression from the wild type promoter was seen following IL-1ß stimulation. IL-1ß caused an increase in the expression from the C/EBP and AP-1 mutant constructs but the differences were not significant. No increase in reporter activity was observed following IL-1ß stimulation of the NF-
B mutant construct (Figure 4
).
|
Discussion
We and others have previously shown that an increase in IL-8 production occurs in uterine tissues in association with the onset and progression of labour in women. This study examined the mechanism by which IL-8 expression may be regulated in uterine tissues. The region of the IL-8 promoter used in these studies was truncated at –135 bp. This truncated region contains binding sites for NF-B, AP-1 and C/EBP, and was able to drive reporter gene expression in both unstimulated and IL-1ß stimulated cells. The binding sites in this short region therefore appear to be sufficient for regulation of gene expression.
Mutation of the NF-B binding site produced a significant reduction in gene expression in unstimulated and stimulated conditions. This has been described in several other cell types. In pulmonary epithelial cells, the NF-B site was shown to be essential for stimulation of IL-8 production in response to mycobacteria and mediated by IL-1 (
Wickremasinghe et al., 1999
). In the colon carcinoma cell line Caco-2, mutation of the NF-B binding site did not alter basal promoter activity but prevented stimulation with IL-1ß (
Wu et al., 1997
). Our data suggest that NF-B plays a central role in both basal and IL-1ß-stimulated expression of IL-8 in both the fetal membranes and the cervix.
NF-B is a hetero- or homodimer composed of two members of the
Rel
gene family which includes
RelA
(p65),
c-Rel
,
NF-kB1
(p50),
Rel B
and
NF-kB2
(Baldwin, 1996). In lymphocyte and fibrosarcoma cell lines it has been found that binding of Rel A or c-Rel to the IL-8 promoter is sufficient to activate transcription, with Rel A binding to the NF-B binding site appearing to have the most consistent effect on IL-8 transcription (
Kunsch and Rosen, 1993
).
The results of our study suggest a difference in regulation of basal
IL-8
gene expression between pre- and post-labour amnion cells. In pre-labour cells, as in HOG-1 cells, mutation of the NF-kB binding site resulted in a reduction in basal activity of the promoter. In post-labour cells, however, this mutation did not alter unstimulated activity, although it did prevent up-regulation of expression by IL-1ß. It is possible that, in post-labour cells, several alternate transcription factors may be available to drive basal IL-8 expression so that mutation of only one transcription factor binding site does not suppress basal promoter activity. Alternatively, it could be that after the onset of labour there is a change in the NF-B subunits available which form more weakly active dimers. Stimulation with IL-1ß may then increase the availability of strongly binding dimers in the tissue. It is also possible that there may be changes in the activity of repressor transcription factors such as Oct-1 (
Wu et al., 1997
). After the onset of labour there may also be increases in the activity of other transcription factors which act on sites other than the NF-B binding site.
Our data suggest that in amnion cells intact C/EBP and AP-1 binding sites are not critical for either basal or stimulated
IL-8
gene expression. In contrast, in the immortalized epithelial cell line HOG-1, mutation of the AP-1 or C/EBP sites significantly reduced basal promoter activity. The close proximity of the C/EBP and the NF-B binding sites enables an interaction between these two transcription factors, allowing synergistic activation of the promoter, as has been shown to occur in non-uterine cell types (Stein and Baldwin, 1993;
Kunsch et al., 1994
;
Roebuck, 1999
). At term, there may be an increased availability of NF-B in the amnion cells, reducing the requirement for an intact C/EBP or AP-1 binding site for constitutive activity. This could be due to alterations in the cell environment with increased inflammatory stimulation. Mutation of the AP-1 and C/EBP sites still allowed some stimulation by IL-1ß in amnion cells, though the increases were not generally significant. The proposed increase in availability of NF-B in amnion cells at term, while reducing the need for other transcription factors to promote basal activity, may not be sufficient to allow significant stimulated activity in the absence of an intact AP-1 or C/EBP site.
We have recently described the importance of an intact NF-B binding site for stimulation of
COX-2
gene expression in amnion cells (
Allport et al., 2000
). Labour is proposed to occur due to a cascade of inflammatory mediators. We have now shown that NF-B is an essential for transcriptional up-regulation of two of the genes whose increase is associated with labour,
COX-2
and
IL-8
. NF-kB therefore appears to play a central role in the onset of human parturition.
Acknowledgements
HOG-1 cells were a kind gift from Dr John White. This work was funded by Tommy's Campaign.
Notes
3 To whom correspondence should be addressed. E-mail: c.elliott{at}ic.ac.uk 
References
Allport, V.C., Slater, D.M., Newton, R. and Bennett, P.R. (2000) NF-kappa B and AP-1 are required for cyclo-oxygenase-2 expression in amnion epithelial cell line (WISH). Mol. Hum. Reprod., 6, 561–565
Barclay, C.G., Brennand, J.E., Kelly, R.W. et al. (1993) Interleukin-8 production by the human cervix. Am. J. Obstet. Gynecol., 169, 625–632.[ISI][Medline]
Baggliolini, M., Walz, A. and Kunkel, S.L. (1989) Neutrophil-attracting peptide-1/interleukin-8: a novel cytokine that attracts neutrophils. J. Clin. Invest., 84, 1045–1049.
Baldwin, A.S. Jr (1996) The NF-kB and IkB proteins: new discoveries and insights. Annu. Rev. Immunol., 14, 649–681.[ISI][Medline]
Bennett, P.R., Rose, M.P., Myatt, L. and Elder, M.G. (1987) Preterm labor: stimulation of arachidonic acid metabolism in human amnion cells by bacterial products. Am. J. Obstet. Gynecol., 156, 649–655.[ISI][Medline]
Elliott C.L., Kelly R.W., Critchley H.O.D. et al. (1998) Regulation of interleukin-8 production in the term human placenta during labor and by antigestagens. Am. J. Obstet. Gynecol., 179, 215–20.[ISI][Medline]
Elliott, C.L., Slater, D.M., Dennes, W. et al. (2000) Interleukin-8 expression in human myometrium: changes in relation to labour onset and with gestational age. Am. J. Reprod. Imunol., 43, 272–277.
Gleeson, R.P., Ayub, M., Wright, J.T. et al. (1990) Fatty acid control of growth of human cervical and endometrial cancer cells Br. J. Cancer, 61, 500–503.[ISI][Medline]
Junqueira, L.C.U., Zugaib, M., Montes, G.S. et al. (1980) Morphological and histochemical evidence for the occurrence of collagenolysis and for the role of neutrophilic polymorphonuclear leucocytes during cervical dilatation. Am. J. Obstet. Gynecol., 138, 273–281.[ISI][Medline]
Kelly, R.W., Leask, R. and Calder, A.A. (1992) Choriodecidual production of interleukin-8 and mechanism of parturition. Lancet, 339, 776–777.[ISI][Medline]
Kunsch, C. and Rosen, C.A. (1993) NF-kB subunit-specific regulation of the interleukin-8 promoter. Mol. Cell. Biol., 13, 6137–6146.
Kunsch, C., Lang, R.K., Rosen, C.A. et al. (1994) Synergistic transcriptional activation of the IL-8 gene by NF-kappa B p65 (Rel A) and NF-IL-6. J. Immunol., 153, 153–164.[Abstract]
Mukaida, N., Shiroo, M., Matsushima, K. (1989) Genomic structure of the human monocyte derived neutrophil chemotactic factor IL-8. J. Immunol., 143, 1366–1371.[Abstract]
Osmers, R., Rath, W., Adelmann-Grill, B.C. et al. (1992) Origin of cervical collagenase during parturition. Am. J. Obstet. Gynecol., 166, 1455–1460.[ISI][Medline]
Roebuck K.A. (1999) Regulation of interleukin-8 gene expression J. Interferon Cytokine Res., 19, 429–438.
Stein, B. and Baldwin, A.S. Jr (1993) Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B. Mol. Cell. Biol., 13, 7191–7198.
Thomson, A.J., Telfer, J.F., Young, A et al. (1999) Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process. Hum. Reprod., 14, 229–236.
Wickremasinghe, M.I., Thomas, L.H. and Friedland, J.S. (1999) Pulmonary epithelial cells are a source of IL-8 in the response to Mycobacterium tuberculosis: essential role of IL-1 from infected monocytes in a NF-kappa B-dependent network. J. Immunol., 163, 3936–3947.
Winkler, M., Fischer, D-C., Hlubeck, M. et al. (1998) Interleukin-1beta and interleukin-8 concentrations in the lower uterine segment during parturition at term. Obstet. Gynecol., 91, 945–949.[Abstract]
Winkler, M., Fischer, D-C., Ruck, P. et al. (1999) Parturition at term: parallel increases in interleukin-8 and proteinase concentrations and neutrophil count in the lower segment. Hum. Reprod., 14, 1096–1100.
Wu, G.D., Lai, E.J., Huang, N. et al. (1997) Oct-1 and CCAAT/enhancer-binding orotein (C/EBP) bind to overlapping elements within the interleukin-8 promoter. J. Biol. Chem., 27, 2396–2403.
Submitted on March 5, 2001; accepted on May 16, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  W. E Ackerman IV, T. L.S Summerfield, D. D Vandre, J. M Robinson, and D. A Kniss Nuclear Factor-Kappa B Regulates Inducible Prostaglandin E Synthase Expression in Human Amnion Mesenchymal Cells Biol Reprod, January 1, 2008; 78(1): 68 - 76. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. G. Marx, M. J. Wentz, L. B. MacKay, D. Schlembach, H. Maul, C. Fittkow, R. Given, Y. Vedernikov, G. R. Saade, and R. E. Garfield Effects of Progesterone on iNOS, COX-2, and Collagen Expression in the Cervix J. Histochem. Cytochem., June 1, 2006; 54(6): 623 - 639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Terzidou, Y. Lee, T. Lindstrom, M. Johnson, S. Thornton, and P. R. Bennett Regulation of the Human Oxytocin Receptor by Nuclear Factor-{kappa}B and CCAAT/Enhancer-Binding Protein-{beta} J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2317 - 2326. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Slater, S. Astle, N. Woodcock, J. E. Chivers, N. C.J. de Wit, S. Thornton, M. Vatish, and R. Newton Anti-inflammatory and relaxatory effects of prostaglandin E2 in myometrial smooth muscle Mol. Hum. Reprod., February 1, 2006; 12(2): 89 - 97. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M Lindstrom and P. R Bennett The role of nuclear factor kappa B in human labour Reproduction, November 1, 2005; 130(5): 569 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. R. Chapman, I. Smyrnias, D. O. C. Anumba, G. N. Europe-Finner, and S. C. Robson Expression of the GTP-Binding Protein (G{alpha}s) Is Repressed by the Nuclear Factor {kappa}B RelA Subunit in Human Myometrium Endocrinology, November 1, 2005; 146(11): 4994 - 5002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Loor, H. M. Dann, R. E. Everts, R. Oliveira, C. A. Green, N. A. J. Guretzky, S. L. Rodriguez-Zas, H. A. Lewin, and J. K. Drackley Temporal gene expression profiling of liver from periparturient dairy cows reveals complex adaptive mechanisms in hepatic function Physiol Genomics, October 17, 2005; 23(2): 217 - 226. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.A.Z. Loudon, S.R. Sooranna, P.R. Bennett, and M.R. Johnson Mechanical stretch of human uterine smooth muscle cells increases IL-8 mRNA expression and peptide synthesis Mol. Hum. Reprod., December 1, 2004; 10(12): 895 - 899. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. R. Chapman, G. N. Europe-Finner, and S. C. Robson Expression and Deoxyribonucleic Acid-Binding Activity of the Nuclear Factor {kappa}B Family in the Human Myometrium during Pregnancy and Labor J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5683 - 5693. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lappas, M. Permezel, H. M. Georgiou, and G. E. Rice Regulation of Phospholipase Isozymes by Nuclear Factor-{kappa}B in Human Gestational Tissues in Vitro J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2365 - 2372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Yellon, A. M. Mackler, and M. A. Kirby The Role of Leukocyte Traffic and Activation in Parturition Reproductive Sciences, September 1, 2003; 10(6): 323 - 338. [Abstract] [PDF]
|
|
|
|
|
|
J. A.Z. Loudon, C. L. Elliott, F. Hills, and P. R. Bennett Progesterone Represses Interleukin-8 and Cyclo-Oxygenase-2 in Human Lower Segment Fibroblast Cells and Amnion Epithelial Cells Biol Reprod, July 1, 2003; 69(1): 331 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lappas, M. Permezel, and G. E. Rice N-Acetyl-Cysteine Inhibits Phospholipid Metabolism, Proinflammatory Cytokine Release, Protease Activity, and Nuclear Factor-{kappa}B Deoxyribonucleic Acid-Binding Activity in Human Fetal Membranes in Vitro J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1723 - 1729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Lee, V. Allport, A. Sykes, T. Lindstrom, D. Slater, and P. Bennett The effects of labour and of interleukin 1 beta upon the expression of nuclear factor kappa B related proteins in human amnion Mol. Hum. Reprod., April 1, 2003; 9(4): 213 - 218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Watari, H. Watari, T. Fujimoto, H. Yamada, J. Nishihira, J. f. Strauss III, and S. Fujimoto Lipopolysaccharide Induces Interleukin-8 Production By Human Cervical Smooth Muscle Cells Reproductive Sciences, February 1, 2003; 10(2): 110 - 117. [Abstract] [PDF]
|
|
|
|
|
|
S. Cauci, S. Guaschino, D. de Aloysio, S. Driussi, D. De Santo, P. Penacchioni, and F. Quadrifoglio Interrelationships of interleukin-8 with interleukin-1{beta} and neutrophils in vaginal fluid of healthy and bacterial vaginosis positive women Mol. Hum. Reprod., January 1, 2003; 9(1): 53 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lappas, M. Permezel, H. M. Georgiou, and G. E. Rice Nuclear Factor Kappa B Regulation of Proinflammatory Cytokines in Human Gestational Tissues In Vitro Biol Reprod, August 1, 2002; 67(2): 668 - 673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Romero, H. Kuivaniemi, and G. Tromp Functional Genomics and Proteomics in Term and Preterm Parturition J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2431 - 2434. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||