help button home button Endocrine Society Molecular Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
20/11/2999    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Son, D.-S.
Right arrow Articles by Roby, K. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Son, D.-S.
Right arrow Articles by Roby, K. F.
Molecular Endocrinology 20 (11): 2999-3013
Copyright © 2006 by The Endocrine Society

Interleukin-1{alpha}-Induced Chemokines in Mouse Granulosa Cells: Impact on Keratinocyte Chemoattractant Chemokine, a CXC Subfamily

Deok-Soo Son and Katherine F. Roby

Center for Reproductive Sciences (D.S.S., K.F.R), Departments of Molecular and Integrative Physiology (D.S.S.), Anatomy and Cell Biology (K.F.R), University of Kansas Medical Center, Kansas City, Kansas 66160

Address all correspondence and requests for reprints to: Deok-Soo Son, Center of Reproductive Sciences, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7417. E-mail: dson{at}kumc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IL-1 is well known to be involved in the immune system and have a role in ovarian inflammation as well as exhibiting inhibitory effects on steroidogenesis and folliculogenesis. Because multiple aspects of ovarian function have also been shown to involve cytokine/chemokine networks, IL-1{alpha}-induced chemokine gene expression in mouse granulosa cells was investigated. Granulosa cells from immature mice at 28 d of age were cultured with IL-1{alpha} (10 ng/ml). IL-1{alpha} induced abundantly and specifically keratinocyte chemoattractant (KC) chemokine, a CXC subfamily. KC chemokine mRNA and protein were increased 1–2 h after IL-1{alpha} and then gradually decreased. The KC promoter (–701/+30) containing three nuclear factor (NF)-{kappa}B sites was fully responsive to IL-1{alpha}, whereas deletions and mutants of the NF-{kappa}B sites lowered the responsiveness to IL-1{alpha}. The proximal NF-{kappa}B site (–69/–59) played a critical role in regulating IL-1{alpha}-induced KC chemokine promoter activity. Overexpression of the inhibitor of NF-{kappa}B (I{kappa}B) blocked KC promoter activity induced by IL-1{alpha}, whereas overexpression of p65, a component of NF-{kappa}B, increased promoter activity and mRNA of KC chemokine. In addition, FSH did not affect NF-{kappa}B signaling or IL-1{alpha}-induced KC chemokine promoter activity. Within 1–3 h after ip injection of lipopolysaccharide (100 µg/mouse), a product known to stimulate release of IL-1, KC chemokine was localized in the ovary to granulosa cells as well as the thecal-interstitial layer. The results of this study indicate that KC gene is a chemokine induced acutely by IL-1{alpha} via NF-{kappa}B signaling in mouse granulosa cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CYTOKINE NETWORKS ARE involved in folliculogenesis, ovulation, luteinization, and ovarian inflammatory events and modulate these processes (1, 2). Ovarian pathophysiological states such as ovarian cancer (3), autoimmune ovarian disease (4), polycystic ovary syndrome (5), and ovarian hyperstimulation syndrome (6) are closely associated with inflammatory mediators such as cytokines and prostaglandins. IL-1, a proinflammatory cytokine, is localized in various ovarian cell types such as the oocyte, granulosa, and theca cells, and ovarian cancer cells, and is involved in ovulation-associated events, steroidogenesis and the growth of ovarian cancer (1, 7, 8). Also, IL-1 is involved in regulating tissue chemokine expression and leukocyte accumulation (9). The abundant influx of leukocytes into the ovary varies with the stage of the cycle and the leukocytes are thought to have a central role in influencing follicular atresia, ovulation, and luteal function (10, 11) and are potentially involved in ovarian disorders such as premature ovarian failure and polycystic ovary syndrome (12). Local chemoattractants trafficking and accumulating leukocytes into the ovary include mainly a family of small cytokines, also known as chemokines. Chemokines are low-molecular weight proinflammatory cytokines involved in numerous biological processes ranging from hematopoiesis and angiogenesis to the extravasation and tissue infiltration of leukocytes in response to inflammatory agents, tissue damage and bacterial or viral infection (13, 14). In the female reproductive tract, chemokines may play a relevant role in many physiologic and pathologic situations, such as ovulation, menstruation, implantation, cervical ripening, preterm labor, and endometriosis (10). During ovarian inflammatory response, proinflammatory cytokine production such as IL-1 is augmented in granulosa cells (15, 16) and can induce local chemokine synthesis, which in turn may affect ovarian function. Therefore, the present study was designed to assess the effects of IL-1{alpha} on chemokines in granulosa cells obtained from immature intact mice. Results from the present study indicate that IL-1{alpha} induced abundantly and specifically keratinocyte chemoattractant (KC) chemokine mRNA and protein in mouse granulosa cells. The expression of KC chemokine by IL-1{alpha} involved nuclear factor {kappa}B (NF-{kappa}B) signaling and required three {kappa}B sites present in the KC promoter with the proximal {kappa}B site being critical in regulating KC promoter activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IL-1{alpha}-Induced Chemokines in Mouse Granulosa Cells
A microarray containing genes that encode mouse chemokines was employed to assess chemokine expression induced by IL-1{alpha} in granulosa cells. The nomenclature approved by the International Union of Immunological Societies/World Health Organization Subcommittee on Chemokine Nomenclature (17) was used in the present study. Based on the number of amino acids between the first cysteine motif, four subfamilies of chemokines are classified as follows: C, CC, CXC, and CX3C (11). Short (1 h)- and long (24 h)-term effects of IL-1{alpha} in vitro on mouse granulosa cell chemokines were determined. CCL17 was the only constitutive chemokine detectable in granulosa cells and IL-1{alpha} did not affect its expression (Table 1Go). Constitutive CCL17 expression has been described in dermal microvessels of noninflamed human skin (18). In addition, inflammatory cytokines such as IL-1 and TNF{alpha} failed to induce CCL17 mRNA in normal human epidermal keratinocytes (19). IL-1{alpha} increased specifically and abundantly KC chemokine mRNAs, CXCL1, and CXCL2 as CXC subfamily members that are mouse homologs of human growth-regulated oncogene (GRO)-{alpha} and ß, respectively (Table 1Go and Fig. 1AGo). IL-1{alpha}-induced KC chemokine isoforms were confirmed by RT-PCR using specific primers (Fig. 1BGo). CXCL1 chemokine expression was strongly induced 1 h after IL-1{alpha}, and its expression remained elevated 24 h after treatment. CXCL2 expression was also induced 1 h after IL-1{alpha}, but its expression was reduced to control levels at 24 h (Table 1Go and Fig. 1Go). CXCL1 shares a high identity of amino acid sequence (65%) with CXCL2 (Table 2Go and Ref. 20). The promoter of CXCL1 contains three {kappa}B sites, whereas the CXCL2 promoter has only one {kappa}B site (21). Previous studies also indicate that CXCL1 is more potent than CXCL2 and CXCL1 is the chemokine primarily responsive to acute in vivo inflammation (22). Therefore, CXCL1 chemokine was further investigated.


View this table:
[in this window]
[in a new window]
  		Table 1. Effects of IL-1{alpha} on Chemokines in Mouse Granulosa Cells as Determined by Microarray (Unit: Expression Signal on Microarray)

 

Figure 1
View larger version (46K):
[in this window]
[in a new window]
  		Fig. 1. Identification of Chemokines Induced by IL-1{alpha} in Mouse Granulosa Cells

A, IL-1{alpha}-induced KC chemokines by microarray. Primary granulosa cells obtained from ovaries of mice at 28 d of age were incubated with vehicle (Control) or IL-1{alpha} (10 ng/ml) for 1 and 24 h. After isolating total RNAs, biotin-16-labeling of the cRNA target was produced from the cDNA template transcribed reversely from total RNA. Upper and lower arrowheads indicate CXCL1 and CXCL2, respectively. B, IL-1{alpha}-induced KC chemokines observed by microarray were confirmed by RT-PCR. Primary granulosa cells were incubated with vehicle (Control, C) or IL-1{alpha} ({alpha}, 10 ng/ml) for 1 and 24 h. After isolating total RNAs, RT-PCR was performed using specific primers of KC isoforms with 25 cycles for CXCL1/2 and 30 cycles for L19. L19 was used as an internal housekeeping gene. M, Molecular marker in base pair. Experiments were performed in duplicate and a representative result is shown.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Percentage of Identity of Amino Acid Sequence in Mouse, Rat, Human, and Bovine CXCL1/2/3 Chemokines as Determined by Aligned Sequences (EBLOSUM62) (Unit: % of Identity)

 
Time-Course of IL-1{alpha}-Induced KC Chemokine (CXCL1)
KC chemokine is well known to be an immediate-early gene in BALB/c-3T3 cells and murine peritoneal macrophages (23, 24). This fact is in part supported by a quick induction of KC chemokine mRNA at 1 h after IL-1{alpha} (Table 1Go and Fig. 1Go). To further delineate the response of KC chemokine to IL-1{alpha}, a time-course of KC chemokine induction by IL-1{alpha} was investigated and compared with the expression pattern of c-JUN, a well-established immediate-early gene in mouse granulosa cells (25). IL-1{alpha} increased KC mRNA and protein in mouse granulosa cells to maximum levels within 1 h and then the levels decreased gradually (Fig. 2Go). The KC protein expression pattern was similar to c-JUN, rising to maximum levels within 1–2 h and then gradually decreasing by 6 h after IL-1{alpha} (Fig. 2Go).


Figure 2
View larger version (61K):
[in this window]
[in a new window]
  		Fig. 2. Time-Course of IL-1{alpha}-Induced KC Chemokine mRNA and Protein in Mouse Granulosa Cells

Primary granulosa cells obtained from ovaries of mice at 28 d of age were incubated with IL-1{alpha} (10 ng/ml) for 0, 1, 2, 3, and 6 h. After isolating total RNAs and proteins, Northern and Western blots were performed using KC cDNA probe and antibodies for KC, c-JUN, and ß-actin, respectively. As loading controls, 28S/18S RNA and ß-actin protein were used for Northern and Western blots, respectively. Experiments were performed twice and a representative result is shown. Therefore, data represent mean relative to OD normalized by maximum value (100%) with no statistical analyses.

 
Effects of IL-1{alpha} on KC Promoter
Human GRO (CXCL1) gene expression has been shown to be regulated by activation of NF-{kappa}B in numerous cell types (26, 27, 28, 29, 30, 31, 32). Also, transcriptional control of the KC gene required {kappa}B sites in the KC promoter in macrophage and 3T3 fibroblast cell lines (21). Regulatory mechanisms controlling KC gene expression in ovarian cells are unknown. Because the KC promoter contains three {kappa}B sites (21), we first determined whether the three {kappa}B sites in the mouse KC promoter were necessary in regulating KC gene expression in granulosa cells by deleting {kappa}B sites. In granulosa cells IL-1{alpha} induced approximately 4-fold increase in luciferase activity of KC701LUC, the full-length mouse KC chemokine promoter containing all three {kappa}B sites (Fig. 3AGo). The KC promoters containing deletions of the {kappa}B sites lowered the responsiveness to IL-1{alpha} (Fig. 3AGo). As compared with approximately 4-fold increase for KC701LUC, deletion of one {kappa}B site resulted in approximately 3-fold for KC95LUC and deletion of two {kappa}B sites resulted in approximately 2-fold for KC72LUC (Fig. 3AGo). However, the promoter with deletion of all {kappa}B sites (KC52LUC) was not responsive. These results indicated that each {kappa}B site in the KC promoter may be functional in regulating KC gene expression in granulosa cells. To determine the role of each {kappa}B site in IL-1{alpha} regulation of the KC promoter, mutants of each {kappa}B site in the KC promoter (Fig. 3BGo) were prepared by site-directed mutagenesis using specific {kappa}B site mutant primers. Mutation of any {kappa}B site lowered the responsiveness as compared with KC701LUC (Fig. 3BGo). In particular, the mutation of the proximal {kappa}B site alone (–69/–59, m1) and when in combination with the mutants KC701LUCm3, KC701LUCm2 and KC701LUCm3m2 was not responsive to IL-1{alpha} (Fig. 3BGo). This finding indicated that the proximal {kappa}B site in the KC promoter was essential for regulation of KC promoter activity in granulosa cells and that the other {kappa}B sites further supported induction of KC promoter activity.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3. Effects of IL-1{alpha} on the Luciferase Activity of KC Promoters

A, Effects of IL-1{alpha} on luciferase activity of {kappa}B site deletions of the KC promoter. The KC701LUC (–701/+30) was generated by PCR using a 1.5-kb DNA fragment of the mouse KC gene. The amplified KC DNA fragment was subcloned into the pGL3-basic vector. The {kappa}B site deletion constructs were generated from the KC701LUC with three {kappa}B sites as follows: –95/+30 deletion construct (KC95LUC) with two {kappa}B sites, –72/+30 deletion construct (KC72LUC) with one {kappa}B site and –52/+30 deletion construct (KC52LUC) without {kappa}B sites. B, Effects of IL-1{alpha} on luciferase activity of {kappa}B site mutants of KC promoter. Site-directed mutants were generated from the KC701LUC by using primers with mutant {kappa}B sites as follows: –615/–585 mutant {kappa}B site (KC701LUCm3), –102/–71 mutant {kappa}B site (KC701LUCm2) and –83/–52 mutant {kappa}B site (KC701LUCm1). Additional mutants of {kappa}B sites were generated as follows: –615/–585 and –102/–71 mutants (KC701LUCm3m2), –615/–585 and –83/–52 mutants (KC701LUCm3m1), –102/–71 and –83/–52 mutants (KC701LUCm2m1), and all mutants (KC701LUCm3m2m1). Primary granulosa cells were transfected for 3.5 h with various KC promoters containing luciferase reporter (500 ng/ml). Cells were incubated with or without IL-1{alpha} (10 ng/ml) overnight. The luciferase activity was normalized to total protein concentrations and expressed as a fold increase by comparison to the control. Dark bars indicate significant increase (P ≤ 0.05) when compared with its own control as calculated by the paired Student’s t test. Cross circles indicate {kappa}B site mutants. C, Control; LUC, luciferase. Experiments were performed in triplicate and all data are shown as mean ± SE.

 
Involvement of NF-{kappa}B Signaling in Regulating the KC Chemokine
In previous studies, TNF, another proinflammatory mediator, activated NF-{kappa}B related molecules such as I{kappa}B and p65 in mouse granulosa cells (33). Thus, involvement of NF-{kappa}B signaling in regulating KC gene expression by IL-1{alpha} was investigated in mouse granulosa cells. IL-1{alpha} treatment increased phosphorylation of I{kappa}B, a NF-{kappa}B regulatory protein, within 5 min, decreased at 15 min, increased again after 30 min and slightly decreased at 60 min after IL-1{alpha} treatment (Fig. 4AGo). This fluctuation in phosphorylation of I{kappa}B is a classical phenomenon of NF-{kappa}B signaling that reflects oscillations in nuclear NF-{kappa}B activity by continued cycles of I{kappa}B degradation and synthesis (34). In contrast to phosphorylated I{kappa}B, total I{kappa}B protein was reduced at 5 min after IL-1{alpha} and then gradually recovered by 30–60 min in granulosa cells (Fig. 4AGo). The NF-{kappa}B signal transduction pathway was monitored by assessing effects of IL-1{alpha} on promoter activity of NF-{kappa}B (-GGGAATTTCC-). IL-1{alpha} increased the luciferase activity of pNF-{kappa}B-Luc in granulosa cells approximately 5-fold (Fig. 4BGo). Thus, effects of I{kappa}B, the NF-{kappa}B inhibitor, and p65, the major active component of NF-{kappa}B, on KC chemokine promoter activity and mRNA expression were assessed. Overexpression of I{kappa}B inhibited basal and IL-1{alpha}-induced luciferase activity of the KC promoter, KC701LUC, in granulosa cells (Fig. 4CGo). Overexpression of p65 increased luciferase activity of the KC701LUC promoter (Fig. 4DGo) and increased KC mRNA (Fig. 4EGo) in granulosa cells.


Figure 4
View larger version (36K):
[in this window]
[in a new window]
  		Fig. 4. Involvement of NF-{kappa}B Signaling in Regulating KC Chemokine Promoter Activity and mRNA in Mouse Granulosa Cells

A, Effects of IL-1{alpha} on I{kappa}B activation. Primary granulosa cells obtained from ovaries of mice at 28 d of age were incubated with IL-1{alpha} (10 ng/ml) for 0, 5, 15, 30, and 60 min. After isolating total proteins, Western blots were performed using antibodies for phospho-I{kappa}B, I{kappa}B and ß-actin. As a loading control, ß-actin was used. Experiments were performed in duplicate and a representative result is shown. B, Effects of IL-1{alpha} on the luciferase activity of the NF-{kappa}B promoter (pNF-{kappa}B-luc vector) containing four tandem copies of the NF-{kappa}B consensus sequence. Primary granulosa cells were transfected for 3.5 h with pNF-{kappa}B-luc vector (500 ng/ml). Cells were incubated with or without IL-1{alpha} (10 ng/ml) overnight. C, Effects of I{kappa}B expression vector on IL-1{alpha}-induced KC promoter activity. Primary granulosa cells were cotransfected for 3.5 h with the KC promoter (KC701LUC, 500 ng/ml) and either empty (100 ng/ml) or I{kappa}B (100 ng/ml) expression vectors. Where indicated cells were incubated with or without IL-1{alpha} (10 ng/ml) overnight. D, Effects of p65 expression vector on KC promoter activity. Primary granulosa cells were cotransfected for 3.5 h with the KC promoter (KC701LUC, 500 ng/ml) and either empty (10 ng/ml) or p65 (10 ng/ml) expression vectors. And then cells were incubated overnight. The luciferase activity was normalized to total protein concentrations and expressed as a fold change by comparison to the control. A dark bar indicates significant increases (P ≤ 0.05) when compared with control as calculated by the paired Student’s t test. Different letters indicate the significant difference (P ≤ 0.05) between groups as analyzed by Tukey’s pairwise comparisons. C, Control; LUC, luciferase. Experiments were performed in triplicate and all data are shown as mean ± SE. E, Effects of p65 expression vector on KC mRNA. Primary granulosa cells were transfected for 3.5 h with empty (10 ng/ml) or p65 (10 ng/ml) expression vectors. After transfection, cells were incubated overnight to allow p65 expression. After isolating total RNAs, RT-PCR was performed using specific primers for KC chemokine with 25 cycles for KC and 30 cycles for L19. The L19 was used as a loading control. M, Molecular marker in base pair. Experiments were performed in duplicate and a representative result is shown.

 
Comparison of CXCL1, 2, and 3 Chemokines among Species and Effects of Other NF-{kappa}B Signaling Activators on the KC Promoter
The proximal {kappa}B site in the KC promoter appeared to be essential for its IL1-{alpha}-induced activation (Fig. 3BGo). The transcriptional control of KC gene is distinct from human GRO (CXCL1, 2, 3) genes (21). Therefore, the importance of the proximal {kappa}B site in regulating KC chemokine was investigated by comparing the 5' flanking sequences of the CXCL1, CXCL2, and CXCL3 chemokines from mouse, rat, human, and cow. All of chemokine promoters examined shared conserved proximal {kappa}B sites (Fig. 5AGo). Human (hCXCL1, 2, and 3) and mouse (mCXCL2) promoters contained only one proximal {kappa}B site (Fig. 5AGo). The rat CXCL1 promoter was similar to the mouse CXCL1 with three {kappa}B-like sites, two proximal and one distal (Fig. 5AGo). The bovine CXCL1 promoter contained two proximal {kappa}B sites without a distal site, whereas the CXCL2 promoter contained proximal and distal sites (Fig. 5AGo). Despite distinct differences of promoters among species, CXCL1, 2, and 3 chemokines shared 50–90% identity in amino acid sequences between and within species (Table 2Go). Within species, human and bovine CXCL1/2/3 chemokines shared higher identity (above 70%) than mouse and rat isoforms (below 70%) (Table 2Go). The conserved proximal {kappa}B site of KC chemokine promoters between species suggests that this site may play a primary role in regulating KC gene expression, whereas other {kappa}B sites may be supportive or secondary. Based on the importance of {kappa}B sites in regulating KC chemokine, all activators of NF-{kappa}B signaling may be candidates to induce KC chemokine in granulosa cells. Our previous studies demonstrated that TNF is an activator of NF-{kappa}B signaling in mouse granulosa cells (33). TNF treatment also exhibited a pattern similar to IL-1{alpha} in increasing KC chemokine promoter activity (Fig. 5BGo). To determine whether the responsiveness to KC chemokine expression was dependent on the activation state, or maturity of granulosa cells, the ability of IL-1{alpha} and TNF to induce KC chemokine promoter activity was compared in granulosa cells isolated from immature mice [no equine chorionic gonadotropin (eCG)] and eCG-primed mice (48 h after eCG). Basal KC promoter activity was not different in granulosa cells from immature mice and granulosa cells collected from immature mice 48 h after eCG. In addition, the ability of IL-1{alpha} and TNF to induce KC promoter activity was not different in the immature and eCG-primed granulosa cells (Fig. 5BGo). These data indicate that the ability of IL-1{alpha} to regulate granulosa cell KC chemokine is not related to the maturation and/or activation state of granulosa cells. To further determine whether physiological activation of the granulosa cells resulted in activation of KC chemokine, the effects of FSH treatment in vitro were assessed. FSH treatment in vitro did not alter KC chemokine promoter activity in granulosa cells and furthermore IL-1{alpha} induced promoter activity was not altered by the addition of FSH (Fig. 5CGo). Similarly, NF-{kappa}B promoter activity in granulosa cells was not affected by FSH and FSH did not alter IL-1{alpha}-induced NF-{kappa}B promoter activity. As anticipated, FSH treatment did induce cAMP-responsive element (CRE) promoter activity in mouse granulosa cells (Fig. 5CGo).


Figure 5
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5. Comparison of CXCL1/2/3 Chemokines among Species and Effects of Other NF-{kappa}B Signaling Activators on the KC Promoter

A, Schematic representation of {kappa}B sites between species in CXC1, 2 and 3 chemokine promoters. DNA sequences in promoters were analyzed with EBLOSUM62 program provided by European Bioinformatics Institute (www.ebi.ac.uk). mCXCL, Mouse CXCL chemokine; rCXCL, rat CXCL chemokine; hCXCL, human CXCL chemokine; bCXCL, bovine CXCL. Accession numbers are as follows: mCXCL1, S79767; mCXCL2, S61346; rCXCL1, D11445; rCXCL2, NW047424; hCXCL1, U03018; hCXCL2, U03019; hCXCL3, U03020; bCXCL1 and bCXCL2, NW616958. Gray circles indicate {kappa}B-like sites. B, Effects of IL-1{alpha} and TNF on KC promoter (KC701LUC) in granulosa cells from intact and eCG-treated mice. Mice at 26 d of age were given vehicle (saline) or eCG (2.5 IU, sc). Ovaries were collected from mice on d 28 and granulosa cells were cultured. Granulosa cells were transfected for 3.5 h with various promoters containing luciferase reporter (500 ng/ml). Cells were incubated with or without IL-1{alpha} and TNF (10 ng/ml) overnight. C, Effects of FSH on the CRE (pCRE-luc), NF-{kappa}B (pNF-{kappa}B-luc vector) and KC (KC701LUC) promoters in granulosa cells from intact and eCG-treated mice. Cells were incubated with FSH (50 ng/ml) in the presence or the absence of IL-1{alpha} (10 ng/ml) overnight. The luciferase activity was normalized to total protein concentrations and expressed as a fold increase by comparison to the control. Dark gray bars indicate significant increase (P ≤ 0.05) between groups as analyzed by Tukey’s pairwise comparisons. C, Control; LUC, luciferase. Experiments were performed in triplicate, and all data are shown as mean ± SE.

 
Ovarian Localization of KC Chemokine after Endotoxin Treatment
Lipopolysaccharide (LPS), a bacterial endotoxin, is well established as a mediator of inflammation, is often used as a tool to initiate inflammatory cascades, and is known to induce release of proinflammatory cytokines such as IL-1 in the ovary (35). Therefore, ovarian localization of KC chemokine by in situ hybridization was assessed after systemic inflammation induced by LPS injection. The ovary from intact, non-LPS-treated animals did not exhibit any positive hybridization (Fig. 6Go, 0 h). KC chemokine mRNA hybridization was detected in the ovary after LPS administration in an intense, yet transient manner; expression was intense at 1 and 2 h after LPS, reduced by 3 h after LPS and was further reduced to nondetectable or basal levels by 6 h after LPS injection (Fig. 6Go). At 1 h after LPS, KC chemokine mRNA was localized to the thecal-interstitial layer with only minimal hybridization detected in the granulosa cell layer (Fig. 6Go; 1 h). Increased expression of KC chemokine in the granulosa cell compartment was observed by 2 h after LPS and a continuous gradient of labeling index from the peripheral granulosa (higher expression) to the center of the follicle (lower expression) was observed (Fig. 6Go; 2 h). Expression was maintained in both the thecal-interstitial and granulosa cell compartments after 3 h, although overall hybridization signals were reduced (Fig. 6Go; 3 h). By 6 h after LPS KC chemokine hybridization was reduced to the levels observed in the control treated ovaries (Fig. 6Go; 6 h). Oocytes did not express KC chemokine mRNA in either vehicle- or LPS-treated animals at any time point examined (Fig. 6Go).


Figure 6
View larger version (105K):
[in this window]
[in a new window]
  		Fig. 6. Ovarian Localization of KC Chemokine after LPS Treatment

Mice at 28 d of age were given vehicle (saline) or LPS (100 µg/mouse, ip). Ovaries were collected at 0, 1, 2, 3, and 6 h after LPS treatment and ovarian localization of KC chemokine was investigated by using in situ hybridization with a DIG-labeled RNA probe. Sense probe of KC chemokine did not express any signal (data not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The main findings in the present study were 1) KC chemokines (CXCL1 and 2) were chemokines induced by IL-1{alpha} in mouse granulosa cells (Table 1Go and Fig. 1Go); 2) all three {kappa}B sites in the KC promoter were required for full induction of KC chemokine by IL-1{alpha}; 3) the proximal NF-{kappa}B site (–69/–59) was critical in regulating any response of the KC promoter to IL-1{alpha} (Fig. 3Go); 4) NF-{kappa}B signaling molecules I{kappa}B and p65 were involved in regulating KC chemokine (Figs. 4Go and 5Go); 5) KC chemokine basal and IL-1{alpha} stimulated promoter activity was not different in immature and eCG-primed granulosa (Fig. 5Go); 6) FSH had no effect on KC chemokine promoter activity (Fig. 5Go); and 7) after LPS administration, KC chemokine was expressed throughout the whole ovary but not in oocytes (Fig. 6Go).

Limited information currently exists regarding chemokines present in the mouse ovary. In an RT-PCR screen, Zhou et al. (36) demonstrated the presence of several chemokines in the mouse ovary including MCP-1 and RANTES. However, KC chemokines were not investigated in that screen. That study also indicated the majority of chemokines detected in the mouse ovary were from peripheral blood leukocytes present in the ovary (36). We also found that analysis by RT-PCR of whole ovary revealed a consistent level of KC chemokine mRNA in the ovary at multiple times during eCG- and human chorionic gonadotropin (hCG)-stimulated follicle development and ovulation, and this expression was likely due to leukocytes in the ovary (unpublished data). As the present study indicates, KC chemokine was expressed by granulosa cells and expression could be regulated by IL1{alpha}, TNF, and LPS, but not FSH. Thymus-expressed chemokine (TECK), a chemokine involved in T-cell development, and its receptor (TECKR) has recently been shown to be present in the mouse ovary (37). TECK expression peaked 10–12 h after an ovulatory dose of hCG and was hypothesized to play a role in the recruitment of mononuclear cells into the ovary around the time of ovulation. The presence of several chemokines have been demonstrated in the human ovary. GRO{alpha}, the homolog of KC chemokine, is produced by granulosa-lutein cells (38) and GRO{alpha} production was induced by IL-1{alpha}, TNF, and cAMP. GRO{alpha} was also present in follicular fluid, and in one study was shown to increase after hCG (39); however, in another study GRO{alpha} levels did not correlate with follicular fluid levels of progesterone and estradiol (40). Using in situ hybridization, we were unable to detect KC chemokine expression in the mouse ovary after eCG- and hCG-stimulated follicle development and ovulation (our unpublished data). However, the finding that KC chemokine was rapidly induced in vivo after LPS administration indicates that KC chemokine may play a role during systemic infection or pathological conditions where chronic inflammation exists.

Induction of KC chemokine expression was rapid, within 1 h after in vitro IL-1{alpha} and in vivo LPS as previously described in various cell types (23, 24). Because mouse granulosa cells expressed c-Jun as an immediate-early gene in response to proinflammatory cytokines such as TNF (25), the expression pattern between KC chemokine and c-Jun was compared and the similarity confirmed KC chemokine as an immediate-early gene in mouse granulosa cells (Fig. 2Go).

To date, there is no information on the mechanisms regulating KC gene expression in ovarian granulosa cells. From analysis of the KC promoter (Fig. 3Go), all three {kappa}B sites were required cooperatively to fully induce KC chemokine in granulosa cells. Induction of the KC gene by LPS in a macrophage cell line and by TNF in 3T3 fibroblasts required cooperation between two {kappa}B sites (21). This subtle difference in regulating KC gene in macrophage and fibroblasts compared with granulosa cells may be due to different stimuli- and/or cell-specific responses as described by other authors (41, 42). The proximal {kappa}B site (–69/–59) appeared to be critical in regulating KC promoter activity (Fig. 3BGo). When comparing a variety of CXCL1/2/3 promoters between species and across isoforms, the proximal {kappa}B site is identical (Fig. 5AGo). The fact that this site is conserved indicated it is likely to serve an important role in regulating expression of the CXCL1/2/3 genes. Because mutations of other {kappa}B sites resulted in reduced responsiveness to IL-1{alpha} and mutation of the proximal site resulted in loss of responsiveness (Fig. 3BGo), the two {kappa}B sites seem to support the proximal {kappa}B site to fully induce KC gene. Analysis of the human GRO promoter revealed that, in addition to the NF-{kappa}B element, the immediate upstream region including CCAAT-enhancing binding protein (32), HMG(I)Y and Sp1 (29, 31) elements were necessary for basal expression of GRO.

In a previous study using rat whole ovarian dispersate cultures, FSH stimulated the secretion of CXCL1 (43) and cAMP increased GRO{alpha} in culture media from human granulosa-lutein cells (38). In addition to three {kappa}B sites, the mouse KC chemokine 5' flanking region contained an activating protein-1, two CCAAT-enhancing binding protein sites, an activating protein-2 and a serum response element (21). The presence of these sites indicates the potential for contribution of hormonal regulation of KC chemokine in granulosa cells, as seen in the rat and human studies (38, 43). However, in mouse granulosa cells FSH did not affect KC promoter activity and the basal level of KC promoter activity was not different in granulosa cells collected for immature mice and 48 h after eCG (Fig. 5Go). This apparent difference in the regulation of chemokine expression may be due to variation between species and/or cell types.

The importance of {kappa}B sites in the KC promoter strengthens the link for involvement of NF-{kappa}B signaling in IL-1{alpha}-induced KC gene in mouse granulosa cells because IL-1{alpha} phosphorylated I{kappa}B, and overexpression of I{kappa}B blocked KC promoter activity and p65 increased the KC promoter activity (Fig. 4Go). In addition, the finding that p65 transfection increased KC mRNA (Fig. 4EGo) confirmed KC chemokine as a target gene of the NF-{kappa}B signal transduction pathway (44). Because TNF activates NF-{kappa}B signaling via a rapid nuclear translocation of p65 protein in mouse granulosa cells (33), TNF was compared with IL-1{alpha} in increasing KC chemokine promoter activity. The similar induction pattern between TNF and IL-1{alpha} indicates KC chemokine may be induced by multiple activators of NF-{kappa}B signaling in mouse granulosa cells (Fig. 5BGo). Furthermore, the finding that granulosa cells between intact and eCG-treated animals showed no difference in IL-1{alpha}- and TNF-induced KC chemokine promoter activity (Fig. 5BGo) indicates KC chemokine expression is regulated by NF-{kappa}B signaling independent of FSH signaling. In Sertoli cells, FSH increased NF-{kappa}B DNA binding activity (45). However, the relationship between NF-{kappa}B and cAMP signaling is poorly understood and will require further investigation. The present data indicate that the NF-{kappa}B pathway functions independent of the cAMP pathway with regard to KC chemokine.

KC chemokine was expressed in granulosa and theca-interstitial layers in the mouse ovary after LPS injection. LPS induces an inflammatory reaction, at least in part through the induction of IL-1 (1, 8, 35). KC chemokine expression was transient, increasing within 1 h and decreasing by 6 h after LPS. Previous studies indicate that, after LPS administration, ovarian function is inhibited; follicle development, ovulation, and luteal function are all reduced (46, 47, 48, 49). LPS administration in heifers resulted in delayed follicle development and ovulation (49). In rats, LPS treatment induced macrophage infiltration and follicular atresia (48). LPS directly induced atresia of bovine follicle in vitro (46) and inhibited estradiol production by rat granulosa cells in vitro (47). KC chemokine-positive hybridization was localized to granulosa and theca-interstitium. It is likely that at least part of the hybridization observed occurred in nonsteroidogeneic cells such as macrophages. Cell-specific localization/identification of hybridization signals in nonsteroidogenic cells awaits future investigation. The association of LPS and KC chemokine with subsequent ovarian function is yet to be determined. However, because KC chemokine expression was early and transient and has been shown to induce leukocyte attraction, it is possible the ovarian KC chemokine expression may be responsible for the infiltration of leukocytes as observed in other systems.

The functional roles of KC chemokine in ovarian granulosa cells are unknown. A primary biological function of KC chemokine is regulating leukocyte traffic including migration (50, 51, 52). KC chemokine has also been shown to affect cell proliferation (53, 54), cell invasion (55), tumor formation (29), and angiogenesis (56). KC chemokine has the potential to impact multiple dynamic events that occur in the ovary including follicular development, ovulation, and luteinization. Whether KC chemokine plays a role in normal ovarian physiology, or in abnormal or pathological conditions within the ovary, will be determined in future studies.

In summary, IL-1{alpha} increased specifically and abundantly KC chemokine in mouse granulosa cells and the NF-{kappa}B signal transduction pathway and three {kappa}B sites in the KC promoter were essential in regulating KC expression. In addition, KC chemokine was expressed rapidly in the theca-interstitial and granulosa layers in the ovary in response to an inflammatory stimulus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Recombinant murine IL-1{alpha} (lyophilized from a 0.2-µm filtered solution in PBS containing 50 µg of BSA per 1 µg of IL-1{alpha}) and recombinant murine TNF (lyophilized from a 0.2-µm filtered solution in PBS containing 50 µg of BSA per 1 µg of TNF) were obtained from R&D Systems (Minneapolis, MN). The following reagents were purchased from Sigma (St. Louis, MO): LPS (from Escherichia coli, 0111:B4), eCG, hCG, penicillin G/streptomycin and fibronectin. Lipofectamine PlusTM, TRIzol, Moloney murine leukemia virus, SP6 and T7 RNA polymerase and all liquid culture media were acquired from Invitrogen (Grand Island, NY). Antisense and sense oligonucleotides of CXCL1 and CXCL2 were obtained from Integrated DNA Technologies (Coralville, IA). Oligo GEArray Microarray for mouse chemokines (OMM-022) and TrueLabeling-AMP Linear RNA Amplication Kit were obtained from SuperArray (Frederick, MD). The KC promoter and its full-length cDNA were kindly provided by Dr. Tom Hamilton (Cleveland Clinic Foundation, Cleveland, OH) and expression plasmid for the NF-{kappa}B component p65-pRc/RSV was from Dr. Tom Maniatis (Harvard University, Cambridge, MA). KC and ß-actin antibodies were purchased from BD Biosciences (Palo Alto, CA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. The phospho-I{kappa}B (Ser 32/36), I{kappa}B and c-JUN antibodies were purchased from Cell Signaling Technology (Beverly, MA). I{kappa}B expression, pNF-{kappa}B-luc and pCRE-luc vectors were obtained from BD Biosciences. The Luciferase Reporter Assay System and the GeneEditor in vitro Site-Directed Mutagenesis System were obtained from Promega (Madison, WI).

Animals
C57BL6 mice from Harlan, Inc. (Indianapolis, IN) were established as breeding colonies in our laboratory. All mice were given commercial pellet feed and drinking water ad libitum and housed with controlled 12-h light, 12-h dark cycle under pathogen-free conditions. Immature mice at 28 d of age were used for granulosa cell culture to exclude the interaction with other signaling cascades as shown in mice under estrous cycle. All handling of animals and procedures conformed to the guidelines set forth by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center.

Cell Cultures and Treatments
Ovaries were collected under a laminar flow hood from mice at 28 d of age and placed in cold DMEM/F12 supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). The ovaries were cleaned of all connective tissues and fat. Follicles were punctured using a 27-gauge needle attached to 1 ml syringe to extrude granulosa cells. Before culture, plates were coated overnight in 4 µg of fibronectin/ml Hanks’ solution. Then, granulosa cells (~2 x 105 cells/ml) were cultured at 37 C in a water-saturated atmosphere of 95% air and 5% CO2 in fibronectin-coated 12- or six-well culture plates with serum-free DMEM/F12 containing penicillin/streptomycin. After overnight culture to allow cellular attachment to the plate, the medium was removed and fresh medium was added. Treatments were initiated as outlined in Results.

Microarray
Total RNA was extracted using using TRIzol reagent according to manufacturer’s instructions. The cDNA template was reverse-transcribed from total RNA using TrueLabeling-AMP Linear RNA Amplication Kit. In vitro-transcription and biotin-16-labeling of the cRNA target were produced from the cDNA and subjected to Oligo GEArray Microarray (OMM-022). Hybridization and detection were performed as follows: prehybridization for 2 h at 60 C, hybridization overnight at 60 C, washing and exposure on x-ray film. Data analysis was conducted using online with GEArray Expression Analysis Suite at www.SuperArray.com. Absolute and comparison analyses were conducted using the following settings: density with average, background with minimum value and normalization with interquartile.

RT-PCR
Total RNA was isolated using TRIzol reagent according to manufacturer’s instructions. The reverse transcriptase reaction conditions, using random primers with Moloney murine leukemia virus, were at 42 C for 60 min followed by 94 C for 10 min. Specific primers were designed as follows: 5'-ATG ATC CCA GCC ACC CGC TCG CTT-3' (sense) and 5'-CCG TTA CTT GGG GAC ACC TTT TAG CAT C-3' (antisense) for CXCL1, 5'-ATG GCC CCT CCC ACC TGC CGG CTC CT-3' (sense) and 5'-GTT AGC CTT GCC TTT GTT CAG TAT CT-3' (antisense) for CXCL2. L19 was used as a control (28). PCR was performed under the following conditions: denaturation at 94 C for 1 min, annealing at 60 C for 1 min and extension at 74 C for 1 min with 25 cycles for CXCL1/2 and 30 cycles for L19. Amplified PCR products were analyzed by electrophoresis in 2% agarose gels containing 1 µg ethidium bromide/ml. The fluorescent images were photographed under UV light.

Northern Blot
Antisense and sense RNA probes were prepared from RT-PCR products after purification and insertion into pGEM-T (Promega) and labeling with digoxigenin (DIG)-uridine triphosphate (Roche, Indianapolis, IN) by in vitro transcription. Total RNA was isolated using TRIzol reagent and 5 µg was separated in agarose-formaldehyde gels, transferred to nylon membranes and crosslinked by UV light. The membranes were subsequently prehybridized and hybridized with KC RNA probe. After hybridization, washing and blocking the membrane were incubated with anti-DIG-AP (alkaline phosphatase) (1:10,000; 30 min), washed incubated with CSPD (disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricycle [3.3.1.1 3, 7]decan}-4-yl) phenyl phosphate; 1:100) and exposed to x-ray film. 28S and 18S rRNAs were visualized under UV light and used as loading controls.

Western Blot
Cell lysates were prepared, fractionated on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes according to established procedures (57). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline Tween 20 [TBST containing 10 mM Tris, 150 mM PBS, 0.05% Tween 20 (pH 8.0)] and incubated with primary antibodies at 1:1000 dilution in blocking solution overnight at 4 C. The membranes were washed three times with TBST followed by incubation for 1 h with horseradish peroxidase-conjugated secondary antibody according to primary antibody used at 1:2500 in 5% milk/TBST. The membranes were then washed and the bands were visualized by enhanced chemiluminescence. After membrane stripping for 10 min with methanol containing 3% H2O2, ß-actin was detected to serve as an internal loading control of cell lysates.

Construction of the KC Promoter-Luciferase Gene
The KC701LUC (–701/+30) was generated by PCR using a 1.5-kb DNA fragment of the mouse KC gene based on a CAT reporter plasmid provided by Dr. Tom Hamilton (Cleveland Clinic Foundation, Cleveland, OH). Primer sets were designed as follows: 5'-TTA GGT ACC CAC AGC TTT CCC GTG GAC TTT-3' for sense containing KpnI site and 5'-TTA CTC GAG GAA CTG GTT AGA GGC TCT GAG-3' for antisense containing XhoI site. The PCR was performed for 35 cycles at 94 C for 1 min, 58 C for 1 min and 74 C for 1 min with a final extension at 74 C for 10 min. The amplified KC DNA fragment was subcloned into the KpnI and XhoI sites of the pGL3-basic vector. Deletion constructs were generated from the KC701LUC under the same PCR conditions using the following primers: 5'-TTA GGT ACC GGT TGC AGG GAA ACA CCC TGT-3' for –95/+30 deleted construct (KC95LUC), 5'-TTA GGT ACC TCC GGG AAT TTC CCT GGC CCG-3 for –72/+30 deleted construct (KC72LUC) and 5'-TTA GGT ACC GGA GCT CTG GAG TTT CGA GCA-3 for –52/+30 deleted construct (KC52LUC). Site-directed mutants were generated from the KC701LUC by using primers with the following mutant {kappa}B sites (underlined lowercase letters): 5'-GAG TTC GGA CTT TCG ccA AGT TCC CAA-3' for –615/–585 mutant {kappa}B site (KC701LUCm3), 5'-CCT TTC CGG TTG CAG ccA AAC ACC CTG TAC TC-3' for –102/–71 mutant {kappa}B site (KC701LUCm2) and 5'-ACA CCC TGT ACT CCG ccA ATT TCC CTG GCC CG-3' for –83/–52 mutant {kappa}B site (KC701LUCm1). Based on these mutants, further mutants of {kappa}B sites were generated as follows: –615/–585 and –102/–71 mutants (KC701LUCm3m2), –615/–585 and –83/–52 mutants (KC701LUCm3m1), –102/–71 and –83/–52 mutants (KC701LUCm2m1), and all mutants (KC701LUCm3m2m1).

Transient Transfection and the Luciferase Assay
Granulosa cells at approximately 50% confluency in 24-well plates were washed once with fresh DMEM/F12 (without additives) and were transiently transfected with vectors for 3.5 h at 37 C using Lipofectamine and Plus solution. Transfected cells were treated as outlined in Results and incubated for 16 h. After rinsing cells with ice-cold PBS and adding lysis buffer (Promega), cell lysates were centrifuged at 12,000 x g for 1 min at room temperature. The supernatant fluid was then used for determination of luciferase activity using a microplate luminometer. Luciferase activity expressed as relative light units was normalized to the protein level.

In Situ Hybridization
Nonradioactive methods for in situ hybridization on frozen sections were performed. Briefly, sections were dried and fixed in 4% paraformaldehysde in diethylpyrocarbonate-PBS, washed in PBS, and treated with 50 µg/ml Proteinase K at room temperature for 10 min. Slides were dipped 0.1 M Triethanolamine-HCl (pH 8.0) and acetic anhydride was added to a final concentration of 0.25% with stirring and the slides were left undisturbed for 10 min followed by washing in diethylpyrocarbonate-PBS. Slides were prehybridized for 3 h at 60 C and hybridized with 1 µg/ml of DIG-labeled KC RNA probes (antisense and sense) as described in Northern blot at 60 C overnight. The next day, slides were washed in 1x, 1.5x, and 2x SCC solution and incubated with 0.2 µg/ml ribonuclease A in 2x saline sodium citrate (SSC) at 37 C for 30 min. Slides were washed in SSC solution, and then finally washed in PBS/BSA/Triton buffer at room temperature for 15 min. Then slides were incubated in 20% sheep serum in PBS/BSA/Triton buffer and then incubated with DIG-antibody coupled to alkaline phosphatase (1:2000) (Roche) at 4 C overnight. Slides were washed three times in PBS/BSA/Triton buffer, washed in AP buffer, and developed with Nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate (Promega) in AP buffer in the dark. Slides were washed in PBS and then coverslips were mounted.

Statistics
Data were analyzed by the paired Student’s t test and one-way ANOVA as appropriate. If statistical significance (P ≤ 0.05) was determined by ANOVA, the data were further analyzed by Tukey’s pairwise comparisons to detect specific differences between treatments.


   ACKNOWLEDGMENTS
 
We thank Dr. Tom Hamilton (Cleveland Clinic Foundation, Cleveland, OH) for the KC promoter and its full-length cDNA, and Dr. Tom Maniatis (Harvard University, Cambridge, MA) for p65-pRc/RSV.


   FOOTNOTES
 
This work was supported by the National Institutes of Health (NIH) Grant P20 RR016475 from the INBRE Program of the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.

Disclosure statement: the authors have nothing to disclose.

First Published Online July 6, 2006

Abbreviations: AP, Alkaline phosphatase; CRE, cAMP- responsive element; DIG, digoxigenin; eCG, equine chorionic gonadotropin; hCG, human chorionic gonadotropin; KC, keratinocyte chemoattractant; LPS, lipopolysaccharide; NF-{kappa}B, nuclear factor-{kappa}B; I{kappa}B, inhibitor of NF-{kappa}B; GRO, growth-regulated oncogene; TECK, thymus-expressed chemokine.

Received for publication January 3, 2006. Accepted for publication June 28, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Terranova PF, Rice VM 1997 Review: cytokine involvement in ovarian processes. Am J Reprod Immunol 37:50–63[Medline]
  2. Wood JR, Strauss III JF 2002 Multiple signal transduction pathways regulate ovarian steroidogenesis. Endocr Metab Disord 3:33–46
  3. Ness RB, Cottreau C 1999 Possible role of ovarian epithelial inflammation in ovarian cancer. J Natl Cancer Inst 91:1459–1467[Abstract/Free Full Text]
  4. Tung KS, Garza KM, Lou Y, Bagavant H 2001 Autoimmune ovarian disease: mechanism of induction and prevention. J Soc Gynecol Investig 8:S49–S51
  5. Amato G, Conte M, Mazziotti G, Lalli E, Vitolo G, Tucker AT, Bellastella A, Carella C, Izzo A 2003 Serum and follicular fluid cytokines in polycystic ovary syndrome during stimulated cycles. Obstet Gynecol 101:1177–1182[Abstract/Free Full Text]
  6. Delvigne A, Rozenberg S 2002 Systematic review of data concerning etiopathology of ovarian hyperstimulation syndrome. Int J Fertil Womens Med 47:211–226[Medline]
  7. Bornstein SR, Rutkowski H, Vrezas I 2004 Cytokines and steroidogenesis. Mol Cell Endocrinol 215:135–141[CrossRef][Medline]
  8. Gerard N, Caillaud M, Martoriati A, Goudet G, Lalmanach AC 2004 The interleukin-1 system and female reproduction. J Endocrinol 180:203–212[Abstract]
  9. Calkins CM, Bensard DD, Shames BD, Pulido EJ, Abraham E, Fernandez N, Meng X, Dinarello CA, McIntyre Jr RC 2002 IL-1 regulates in vivo C-X-C chemokine induction and neutrophil sequestration following endotoxemia. J Endotoxin Res 8:59–67[Medline]
  10. Garcia-Velasco JA, Arici A 1999 Chemokines and human reproduction. Fertil Steril 71:983–993[CrossRef][Medline]
  11. Townson DH, Liptak AR 2003 Chemokines in the corpus luteum: Implications of leukocyte chemotaxis. Reprod Biol Endocrinol 1:94[CrossRef][Medline]
  12. Bukulmez O, Arici A 2000 Leukocytes in ovarian function. Hum Reprod Update 6:1–15[Abstract/Free Full Text]
  13. Rottman JB 1999 Key role of chemokines and chemokine receptors in inflammation, immunity, neoplasia, and infectious disease. Vet Pathol 36:357–367[Abstract]
  14. Olson TS, Ley K 2002 Chemokines and chemokine receptors in leukocyte trafficking. Am J Physiol Regul Integr Comp Physiol 283:R7–R28
  15. Simon C, Frances A, Piquette G, Polan ML 1994 Immunohistochemical localization of the interleukin-1 system in the mouse ovary during follicular growth, ovulation, and luteinization. Biol Reprod 50:449–457[Abstract]
  16. Nakamura Y, Tamura H, Takiguchi S, Kashida S, Yamagata Y, Sugino N, Kato H 1999 Changes in interleukin-1beta mRNA expression in the rat ovary during the estrous cycle in response to lipopolysaccharide. Endocr J 46:293–300[Medline]
  17. International Union of Immunological Societies/World Health Organization Subcommittee on Chemokine Nomenclature 2002 Chemokine/chemokine receptor nomenclature. J Interferon Cytokine Res 22:1067–1068[CrossRef][Medline]
  18. Chong BF, Murphy JE, Kupper TS, Fuhlbrigge RC 2004 E-selectin, thymus- and activation-regulated chemokine/CCL17, and intercellular adhesion molecule-1 are constitutively coexpressed in dermal microvessels: a foundation for a cutaneous immunosurveillance system. J Immunol 172:1575–1581[Abstract/Free Full Text]
  19. Tsuda T, Tohyama M, Yamasaki K, Shirakata Y, Yahata Y, Tokumaru S, Sayama K, Hashimoto K 2003 Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro. Dermatol Sci 31:37–42[CrossRef]
  20. Wuyts A, Haelens A, Proost P, Lenaerts JP, Conings R, Opdenakker G, Van Damme J 1996 Identification of mouse granulocyte chemotactic protein-2 from fibroblasts and epithelial cells. Functional comparison with natural KC and macrophage inflammatory protein-2. J Immunol 157:1736–1743[Abstract]
  21. Ohmori Y, Fukumoto S, Hamilton TA 1995 Two structurally distinct kappa B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages. J Immunol 155:3593–3600[Abstract]
  22. Call DR, Nemzek JA, Ebong SJ, Bolgos GR, Newcomb DE, Wollenberg GK, Remick DG 2001 Differential local and systemic regulation of the murine chemokines KC and MIP2. Shock 15:278–284[Medline]
  23. Cochran BH, Reffel AC, Stiles CD 1983 Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell 33:939–947[CrossRef][Medline]
  24. Introna M, Bast Jr RC, Tannenbaum CS, Hamilton TA, Adams DO 1987 The effect of LPS on expression of the early "competence" genes JE and KC in murine peritoneal macrophages. J Immunol 138:3891–3896[Abstract]
  25. Son DS, Arai KY, Roby KF, Terranova PF 2004 Tumor necrosis factor alpha (TNF) increases granulosa cell proliferation: dependence on c-Jun and TNF receptor type-1. Endocrinology 145:1218–1226[Abstract/Free Full Text]
  26. Anisowicz A, Messineo M, Lee SW, Sager R 1991 An NF-{kappa}B-like transcription factor mediates IL-1/TNF-{alpha} induction of GRO in human fibroblasts. J Immunol 147:520–527[Abstract]
  27. Devalaraja MN, Wang DZ, Ballard DW, Richmond A 1999 Elevated constitutive I{kappa}B kinase activity and I{kappa}B-{alpha} phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-{alpha} transcription. Cancer Res 59:1372–1377[Abstract/Free Full Text]
  28. Loukinova E, Chen Z, Van Waes C, Dong G 2001 Expression of proangiogenic chemokine Gro 1 in low and high metastatic variants of Pam murine squamous cell carcinoma is differentially regulated by IL-1{alpha}, EGF and TGF-ß1 through NF-{kappa}B dependent and independent mechanisms. Int J Cancer 94:637–644[CrossRef][Medline]
  29. Luan J, Shattuck-Brandt R, Haghnegahdar H, Owen JD, Strieter R, Burdick M, Nirodi C, Beauchamp D, Johnson KN, Richmond A 1997 Mechanism and biological significance of constitutive expression of MGSA/GRO chemokines in malignant melanoma tumor progression. J Leukoc Biol 62:588–597[Abstract]
  30. Shattuck RL, Wood LD, Jaffe GJ, Richmond A 1994 MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells. Mol Cell Biol 14:791–802[Abstract/Free Full Text]
  31. Wood LD, Farmer AA, Richmond A 1995 HMGI(Y) and Sp1 in addition to NF-{kappa}B regulate transcription of the MGSA/GRO{alpha} gene. Nucleic Acids Res 23:4210–4219[Abstract/Free Full Text]
  32. Wood LD, Richmond A 1995 Constitutive and cytokine-induced expression of the melanoma growth stimulatory activity/GRO{alpha} gene requires both NF-{kappa}B and novel constitutive factors. J Biol Chem 270:30619–30626[Abstract/Free Full Text]
  33. Son DS, Roby KF, Terranova PF 2004 Tumor necrosis factor alpha (TNF) induces serum amyloid A3 in mouse granulosa cells. Endocrinology 145:2245–2252[Abstract/Free Full Text]
  34. Ting AY, Endy D 2002 Decoding NF-{kappa}B signaling. Science 298:1189–1190[Abstract/Free Full Text]
  35. De SK, McMaster MT, Andrews GK 1990 Endotoxin induction of murine metallothionein gene expression. J Biol Chem 265:15267–15274[Abstract/Free Full Text]
  36. Zhou C, Borillo J, Wu J, Torres L, Lou YH 2004 Ovarian expression of chemokines and their receptors. J Reprod Immunol 63:1–9[CrossRef][Medline]
  37. Zhou C, Wu J, Borillo J, Torres L, McMahon J, Bao Y, Lou YH 2005 Transient expression of CC chemokine TECK in the ovary during ovulation: its potential role in ovulation. Am J Reprod Immunol 53:238–248[Medline]
  38. Zeineh K, Kawano Y, Fukuda J, Nasu K, Narahara H, Miyakawa I 2003 Possible modulators of IL-8 and GRO-{alpha} production by granulosa cells. Am J Reprod Immunol 50:98–103[CrossRef][Medline]
  39. Oral E, Seli E, Bahtiyar MO, Jones EE, Arici A 1997 Growth-regulated {alpha} expression in human preovulatory follicles and ovarian cells. Am J Reprod Immunol 38:19–25[Medline]
  40. Karstrom-Encrantz L, Runesson E, Bostrom EK, Brannstrom M 1998 Selective presence of the chemokine growth-regulated oncogene {alpha} (GRO{alpha}) in the human follicle and secretion from cultured granulosa-lutein cells at ovulation. Mol Hum Reprod 4:1077–1083[Abstract/Free Full Text]
  41. Ohmori Y, Hamilton TA 1994 Cell type and stimulus specific regulation of chemokine gene expression. Biochem Biophys Res Commun 198:590–596[CrossRef][Medline]
  42. Armstrong DA, Major JA, Chudyk A, Hamilton TA 2004 Neutrophil chemoattractant genes KC and MIP-2 are expressed in different cell populations at sites of surgical injury. J Leukoc Biol 75:641–648[Abstract/Free Full Text]
  43. Ushigoe K, Irahara M, Fukumochi M, Kamada M, Aono T 2000 Production and regulation of cytokine-induced neutrophil chemoattractant in rat ovulation. Biol Reprod 63:121–126[Abstract/Free Full Text]
  44. Pahl HL 1999 Activators and target genes of Rel/NF-{kappa}B transcription factors. Oncogenes 18:6853–6866
  45. Delfino F, Walker WH 1998 Stage-specific nuclear expression of NF-{kappa}B in mammalian testis. Mol Endocrinol 12:1696–1707[Abstract/Free Full Text]
  46. Bosu WT, Perez GI, Kujjo LL 1996 Natural and endotoxin-induced atresia of preantral and early antral follicles is characterized by DNA internucleosomal cleavage. Mol Reprod Dev 44:352–359[CrossRef][Medline]
  47. Taylor CC, Terranova PF 1996 Lipopolysaccharide inhibits in vitro luteinizing hormone-stimulated rat ovarian granulosa cell estradiol but not progesterone secretion. Biol Reprod 54:1390–1396[Abstract]
  48. Besnard N, Horne EA, Whitehead SA 2001 Prolactin and lipopolysaccharide treatment increased apoptosis and atresia in rat ovarian follicles. Acta Physiol Scand 172:17–25[CrossRef][Medline]
  49. Suzuki C, Yoshioka K, Iwamura S, Hirose H 2001 Endotoxin induces delayed ovulation following endocrine aberration during the proestrous phase in Holstein heifers. Domest Anim Endocrinol 20:267–278[CrossRef][Medline]
  50. Lira SA, Zalamea P, Heinrich JN, Fuentes ME, Carrasco D, Lewin AC, Barton DS, Durham S, Bravo R 1994 Expression of the chemokine N51/KC in the thymus and epidermis of transgenic mice results in marked infiltration of a single class of inflammatory cells. J Exp Med 180:2039–2048[Abstract/Free Full Text]
  51. Tsai WC, Strieter RM, Wilkowski JM, Bucknell KA, Burdick MD, Lira SA, Standiford TJ 1998 Lung-specific transgenic expression of KC enhances resistance to Klebsiella pneumoniae in mice. J Immunol 161:2435–2440[Abstract/Free Full Text]
  52. Lentsch AB, Yoshidome H, Cheadle WG, Miller FN, Edwards MJ 1998 Chemokine involvement in hepatic ischemia/reperfusion injury in mice: roles for macrophage inflammatory protein-2 and KC. Hepatology 27:1172–1177[CrossRef]
  53. Filipovic R, Jakovcevski I, Zecevic N 2003 GRO-{alpha} and CXCR2 in the human fetal brain and multiple sclerosis lesions. Dev Neurosci 25:279–290[CrossRef][Medline]
  54. Rennekampff HO, Hansbrough JF, Woods Jr V, Dore C, Kiessig V, Schroder JM 1997 Role of melanoma growth stimulatory activity (MGSA/gro) on keratinocyte function in wound healing. Arch Dermatol Res 289:204–212[CrossRef][Medline]
  55. Woodward JK, Elshaw SR, Murray AK, Nichols CE, Cross N, Laws D, Rennie IG, Sisley K 2002 Stimulation and inhibition of uveal melanoma invasion by HGF, GRO, IL-1{alpha} and TGF-ß. Invest Ophthalmol Vis Sci 43:3144–3152[Abstract/Free Full Text]
  56. Loukinova E, Dong G, Enamorado-Ayalya I, Thomas GR, Chen Z, Schreiber H, Van Waes C 2000 Growth regulated oncogene-alpha expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC receptor-2 dependent mechanism. Oncogene 19:3477–3486[CrossRef][Medline]
  57. Sambrook J, Fritsch EF, Maniatis T 1989 Detection and analys