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BOR - Papers in Press, published online ahead of print January 19, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.032953
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BIOLOGY OF REPRODUCTION 72, 1169–1176 (2005)
DOI: 10.1095/biolreprod.104.032953
© 2005 by the Society for the Study of Reproduction, Inc.

Cooperative Expression of Monocyte Chemoattractant Protein 1 Within the Bovine Corpus Luteum: Evidence of Immune Cell-Endothelial Cell Interactions in a Coculture System1

Amy R. Liptak 3, Brian T. Sullivan 3, Luiz E. Henkes 4, Missaka P.B. Wijayagunawardane 5, Akio Miyamoto 6, John S. Davis 7, Bo R. Rueda 4, and David H. Townson 2 3

Department of Animal and Nutritional Sciences,3 University of New Hampshire, Durham, New Hampshire 03824 Vincent Center for Reproductive Biology,4 Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114 Department of Animal Science,5 University of Peradeniya, Peradeniya 20400, Sri Lanka Department of Agricultural and Life Science,6 Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan Department of Obstetrics and Gynecology,7 University Nebraska Medical Center and Veterans Affairs Medical Center, Omaha, Nebraska 68198


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF2{alpha} together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF2{alpha} for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P < 0.05). Basal secretion of EDN1 by EC was substantial (~2 ng/ml), but was not affected by coculture with PBMC (P > 0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P < 0.05), but again, EDN1 secretion was unchanged (P > 0.05). Interestingly, PGF2{alpha} did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P > 0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF2{alpha} (P > 0.05), but MLC produced less progesterone in the presence of ActPBMC (P < 0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF2{alpha}. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.

corpus luteum, corpus luteum function, cytokines, immunology, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocyte chemoattractant protein 1 (also known as chemokine ligand 2 or CCL2) is a chemical signal known to attract monocytes and T lymphocytes to sites of inflammation and has been implicated as a chemotactic molecule for the recruitment of immune cells into the corpus luteum (CL), particularly at the time of luteal regression [1–7]. Recent investigations of CCL2 expression in vivo and in vitro have ascertained that endothelial cells (EC) of the CL are a potent source of CCL2, and that EC secretion of CCL2 occurs in direct response to cytokine stimulation, specifically in response to tumor necrosis factor {alpha} (TNF) and interferon {gamma} (IFNG) [5, 7, 8]. Surprisingly, however, the luteolytic agent prostaglandin F2{alpha} (PGF2{alpha}) has no direct effect in vitro on the secretion of CCL2 by EC derived from the CL [8], despite the observation that PGF2{alpha}-induced regression of the CL in vivo is accompanied by an increase in CCL2 mRNA expression [3, 4, 9]. These observations indicate that the effect of PGF2{alpha} to provoke CCL2 expression within the CL is possibly mediated through a variety of cell types and/or cellular factors.

Similar to CCL2, endothelin 1 (EDN1) is produced within the CL in response to PGF2{alpha} and is thought to contribute to the functional and structural aspects of luteal regression [10–15]. It is a 21-amino acid vasoconstrictive peptide originally isolated from cultured porcine aortic endothelial cells [16], but is produced by a variety of cell types, including Sertoli cells, granulosal cells, and luteal cells [17–20]. Similar to CCL2, EDN1 is also regulated by cytokines, including TNF and IFNG [21–23]. The potential interactions between immune cells and cells of the CL, with regard to CCL2 and EDN1 expression, have not been fully explored.

Although the composition of the CL is known to change throughout the luteal phase, a striking characteristic of this tissue is the relative abundance of endothelial cells [24] and the relatively sparse numbers of immune cells present during early development [2, 7, 25, 26]. Coincidentally, it is during this period of early development that the bovine CL is also most resistant to the luteolytic effect of PGF2{alpha} [13, 27, 28]. Perhaps immune cells within the CL mediate PGF2{alpha}-induced expression of CCL2 and EDN1 by endothelial cells and the luteolytic effect of PGF2{alpha} at the end of the estrous cycle and pregnancy. Considering that cytokines directly provoke CCL2 and EDN1 secretion [8, 29], interactions between immune cells and EC of the CL might account for increases in CCL2 and EDN1 expression during PGF2{alpha}-induced luteal regression. The relationship between immune cells and the steroidogenic cells of the CL in this environment may also be significant but has not been carefully examined. In the current study, our objectives were to 1) determine if immune cells and PGF2{alpha} regulate CCL2 and EDN1 expression by EC of the bovine CL and 2) investigate the potential effects of immune cells and PGF2{alpha} on CCL2, EDN1, and progesterone production by steroidogenic cells of the bovine CL.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Culture vessels were purchased from Corning, Inc. (Corning, NY). Endothelial cell growth medium and basal medium (EGM-1 and EBM-2, respectively) were obtained from Cambrex (San Diego, CA). RPMI 1640, Histopaque-1083, prostaglandin F2{alpha}-Tris salt, Tri reagent, ITS (insulin, transferrin, selenium), and concanavalin A were obtained from Sigma (St. Louis, MO). Ham F-12 was purchased from GibcoBRL (Grand Island, NY). Bovine recombinant IFNG was a generous gift of Dr. Dale Godson (Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, Canada). Primer sequences for bovine IFNG and glyceraldehyde phosphate dehydrogenase (GAPDH) were obtained from Sigma Genosys (The Woodlands, TX). Reverse transcription-polymerase chain reaction (RT-PCR) thin-wall tubes were purchased from MJ Research (Watertown, MA). GeneAmp Gold RNA PCR Kit was obtained from Applied BioSystems (Atlanta, GA). Gentamicin and the low-mass molecular-weight marker were obtained from Invitrogen (Carlsbad, CA). The EDN1 enzyme immunoassay (EIA) polyclonal antibody was obtained from Peptide Institute, Inc. (Osaka, Japan), and secondary antibody from Seikagaku Co. (Tokyo, Japan). The 96-well EIA plates were purchased from Nunc (Roskilde, Denmark). The biotin labeling kit was from Boehringer Mannheim (Mannheim, Germany), the streptavidin-peroxidase was from Sigma, and the 3,3',5,5' tetramethylbenzadine was from Wako Chemical Co. (Osaka, Japan). Hyperfilm and [{alpha}32P] dCTP were obtained from Amersham Pharmacia (Piscataway, NJ) and 18S rRNA from Ambion (Austin, TX). The prostaglandin F2{alpha} receptor (PTGFR) cDNA was synthesized using primer sequences published previously by Mamluk et al. [30]. All remaining reagents and materials were purchased from either Sigma, VWR (Boston, MA), or Fisher Scientific (Pittsburgh, PA).

Animal Procedures

The University of New Hampshire Animal Care and Use Committee approved all of the following experiments (IACUC 020201).

Peripheral Blood Mononuclear Cell Isolation and Culture

Dissociation and isolation procedures to recover adequate numbers of immune cells directly from bovine CL for the proposed coculture experiments were impractical. Instead, bovine peripheral blood mononuclear cells (PBMC) were collected from jugular venous blood of prepubertal heifers 8–12 months of age. Prepubertal heifers were used in these experiments, rather than reproductively mature cows, to optimize conditions for PBMC isolation, yield, and responsiveness in vitro. The PBMC were isolated by differential centrifugation using Histopaque-1083 according to the manufacturer's instructions. The PBMC were cultured in RPMI 1640 with 10% fetal bovine serum (FBS) and 20 µg/ml gentamicin and equilibrated overnight at 37°C, 95% air, 5% CO2 in a humidified incubator. The PBMC were then placed in fresh culture medium and exposed to vehicle or activated with concanavalin A (10 µg/ml; designated as ActPBMC) for 12 h before coculture with EC derived from bovine CL (experiments I and II; see Fig. 1 for coculture design) or with mixed luteal cells (experiments III and IV; see Fig. 1 for coculture design). Activation of the PBMC by concanavalin A was verified by measurement of IFNG mRNA and protein (see Methods below) as described previously [31]. In all experiments, treatments were conducted in triplicate for 48 h. Cell culture conditions and reagent concentrations were based on previous studies [32–34], including a study in which CCL2 secretion by EC was optimized [8].



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  		FIG. 1. Coculture design. Mixed luteal cells, purified endothelial cells (EC), peripheral blood mononuclear cells (PBMC), and concanavalin A-stimulated PBMC (ActPBMC) were cultured alone or in various combinations. Following the incubation period, the supernatants were removed for analysis of the proinflammatory mediators endothelin 1 and monocyte chemoattractant protein 1

Experiments I and II: Endothelial Cell Cultures

Purified endothelial cells from bovine CL of early pregnancy (~Day 60 based on fetal crown-rump length) were obtained from Cambrex Biosciences (BioWhittaker, Inc., Walkersville, MD) as described previously [8]. The cells (passage 6) were seeded 30 000 cells/ml and grown to confluence in 24-well plates for coculture. The EC were cultured in EGM-2 with 3% FBS at 37°C in a humidified incubator of 95% air and 5% CO2. Prior to coculture with PBMC, the EC cultures were transitioned from EGM-2 medium with 3% FBS to RPMI 1640 medium containing 10% FBS over a 5-day period. On Day 6 of culture, PBMC or ActPBMC were added to the EC cultures (100 000 PBMC/well) and cocultured for 48 h (see Fig. 1). Treatment groups consisted of EC, PBMC, and ActPBMC cultured alone, and cocultures of EC+PBMC and EC+ActPBMC exposed to vehicle (control) and PGF2{alpha} (1 µM). Additional controls included random wells of EC alone treated with concanavalin A or with bovine recombinant IFNG (200 IU/ml) as a positive control [8].

Experiments III and IV: Mixed Luteal Cell Cultures

To initially investigate the potential effects of immune cells and PGF2{alpha} on CCL2, EDN1, and progesterone production by steroidogenic cells of the bovine CL, mixed luteal cultures were established to then coculture with PBMC. Briefly, Day 12 bovine CL (Day 0 = standing estrus) were obtained by transvaginal luteectomy of cows and enzymatically dissociated as previously described [35]. The mixed luteal cells (MLC; i.e., consisting primarily of steroidogenic cells and relatively few EC) were seeded at 125 000 steroidogenic cells/ml in 24-well plates and cultured in Ham F-12 with ITS (5 µg/5 µg/5 ng/ml) and gentamicin (20 µg/ml) for 24 h. Similar to the EC cultures described above, the MLC cultures were transitioned from Ham F-12 medium with ITS to RPMI 1640 medium containing 10% FBS over a 3-day period before coculture with PBMC or ActPBMC. On Day 4 of culture, the PBMC and ActPBMC were added to the MLC cultures (100 000 PBMC/well) and cocultured for 48 h (see Fig. 1). As described above, treatments consisted of EC, PBMC, and ActPBMC cultured alone, and corresponding cocultures exposed to vehicle (control) and PGF2{alpha} (1 µM). Again as a positive control, random wells of MLC alone were treated with bovine recombinant IFNG (200 IU/ml) for 48 h.

Lastly, to ascertain the contribution of cell-cell contact between PBMC and EC and between PBMC and MLC in these experiments, EC and MLC were cultured in the presence of supernatants of PBMC- or ActPBMC-conditioned medium. Briefly, PMBC- and ActPBMC-conditioned medium was centrifuged for 10 min at 8000 x g to obtain a cell-free supernatant before adding to either EC or MLC cultures. The supernatants were added to cultures of EC or MLC (1:1, vol:vol) and the cultures continued under conditions identical to those described above for the coculture experiments (see Fig. 1).

Immunoassays

Monocyte chemoattractant protein 1 (CCL2) CCL2 secretion was measured in conditioned culture medium using a DuoSet hMCP1 sandwich ELISA development system (R&D Systems, Minneapolis, MN). The assay has been validated previously for the detection of bovine CCL2 [8, 36]. All samples were assayed in duplicate using 96-well plates. The mean interassay and intraassay coefficients of variation were 12.4% and 7.8%, respectively. Results were expressed as picograms of CCL2 per milliliter of conditioned medium.

Endothelin 1 (EDN1) The enzyme immunoassay for EDN1 was based on a second antibody method and biotin-streptavidin-peroxidase technique [11]. The EDN1 peptide was labeled with D-biotinol-{varepsilon}-aminocaproic-N-hydroxysuccinimide ester (biotin-7-NHS) with a molecular ratio of 1:2. Duplicates of 15 µl standard or unknown samples in EIA buffer (42 mM Na2HPO4, 8 mM KH2PO4, 20 mM NaCl, 4.8 mM EDTA, 0.05% BSA, pH 7.5) were incubated with 100 µl polyclonal antibody (1:40 000) in 96-well plates at 4°C for 20 h. The wells were coated with 50 µg of secondary antibody (anti-rabbit IgG) beforehand. Following the 20-h incubation, the plates were decanted and the labeled, biotinyl-EDN1 peptide (1:8000 in 100 µl EIA buffer) was added. Plates were further incubated for 2 h, decanted, and then streptavidin-peroxidase (20 ng in 100 µl EIA buffer) was added. After a 15-min incubation, the plates were decanted again and then immediately washed four times with 300 µl/well Tween 80 (0.05%). The substrate reaction was induced with 0.025% 3,3',5,5' tetramethylbenzadine and stopped by 2 M H2SO4 (50 µl/well). The absorbance was measured at 450 nm with a plate reader and analyzed using Microplate manager software (Bio-Rad Lab, CA). Interassay and intraassay coefficients of variance were 13.2% and 9.6%, respectively, and the range of the standard curve was 125–20 000 pg/ml. The median effective dose (ED50) for the assays was 2000 pg/ml, and the cross-reactivity of EDN1 antiserum with EDN1, EDN2, EDN3, and big endothelin were 100%, 60%, 40%, and <1%, respectively. Results were expressed as picograms of EDN1 per milliliter of conditioned medium.

Interferon {gamma} (IFNG) To verify concanavalin A activation of PBMC (i.e., of Act PBMC), IFNG protein was measured in conditioned culture medium using a bovine IFNG sandwich ELISA from Biocor Animal Health, Inc. (Omaha, NE). The kit was modified to include a standard curve. The specificities of the antibodies in this kit have been validated previously, and their cross-reactivity with bovine interferons alpha and beta is negligible [37, 38]. All samples were assayed in duplicate. The mean interassay and intraassay coefficients of variation were 6.4% and 4.5% respectively. Results were expressed as nanograms of IFNG per milliliter of conditioned medium.

Progesterone Progesterone in MLC-conditioned media was measured by radioimmunoassay as previously described [39, 40]. All samples were assayed in duplicate. The mean interassay and intraassay coefficients of variation were 12.8% and 9%, respectively. Results were expressed as nanograms of progesterone per milliliter of conditioned medium.

Reverse Transcriptase-Polymerase Chain Reaction

As an additional measure to verify concanavalin A activation of PBMC, levels of mRNA for IFNG and GAPDH were detected using GeneAmp Gold RNA PCR kit according to the manufacturer's protocol. Total cellular RNA was obtained from preparations of PBMC and ActPBMC immediately before coculture experiments and isolated using Tri Reagent according to the manufacturer's specifications. Approximately 400 ng of total RNA template were used per reverse transcriptase reaction (for IFNG: forward primer, 5'-TATGGCCAGGGCCAATTTTTTAGAGAAATAG-3'; reverse primer, 5'-TACGTTGATGCTCTCCGGCCTCGAAAGAG-3'; and for GAPDH: forward primer, 5'-TGTTCCAGTATGATTCCACCC-3'; reverse primer, 5'-GTCTTCTGGGTGGCAGTGAT-3'). The PCR reactions were performed under the following conditions: 40 cycles at 95°C for 1 min, 60°C for 1 min. PCR products were electrophoretically separated on a 1.5% agarose surface-tension gel and viewed under ultraviolet illumination using the MultiDoc-It Digital Imaging System.

PGF2{alpha} Receptor

Total cellular RNA was isolated from MLC and from PBMC as cells were initially isolated, and from EC upon initial culture, using Tri Reagent in accordance with the manufacturer's instructions. Northern analysis was performed as previously described [8]. Briefly, total RNA (15 µg) was separated by 1.5% agarose:formaldehyde gel electrophoresis and then transferred to nylon membranes by capillary blotting. The RNA was fixed to membranes that were then hybridized overnight with a radiolabeled cDNA probe encoding bPTGFR. Blots were washed and exposed to film. The 18S rRNA was visualized by ethidium bromide to verify loading.

Statistical Analysis

All data were analyzed by analysis of variance (ANOVA) using the general linear model procedure of Minitab (State College, PA) in which effects of experiment, coculture, PGF2{alpha}, and the interaction of coculture with PGF2{alpha} were partitioned. A Tukey test for pairwise comparisons among means of treatment groups was used as a posttest following a significant F-test (P < 0.05). Results are expressed as the mean ± SEM. Experiments I and II were repeated six times using a separate preparation of isolated EC and PBMC for a given experiment (n = 6). Experiments III and IV were repeated three times using a separate CL dissociation and PBMC isolation for a given experiment (n = 3).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Verification of the Activation of PBMC with Concanavalin A

Expression of IFNG was not detectable in PBMCs by RT-PCR (Fig. 2) or by immunoassay of PBMC-conditioned medium. Similarly, IFNG was not detectable in EC or EC-conditioned medium and was not detectable in cocultures of EC+PBMC in five of six experiments (60 ± 60 pg/ml, n = 6). In contrast, PBMC exposed to concanavalin A (designated as ActPBMC) under identical conditions resulted in detectable levels of both IFNG mRNA (Fig. 2) and protein (310 ± 170 pg/ml, n = 6), which confirmed the stimulation of ActPBMC by concanavalin A. The concentration of IFNG in conditioned medium of EC+ActPBMC cocultures (320 ± 220 pg/ml, n = 6) was not different (P > 0.05) than conditioned medium of ActPBMC alone.



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  		FIG. 2. Representative ethidium bromide-stained gel of IFNG (lanes 2 and 4) and GAPDH (lanes 1 and 3) cDNA from peripheral blood mononuclear cells (PBMC) treated with vehicle and concanavalin A (10 µg/ml; ActPBMC). Reverse transcriptase-polymerase chain reaction (RT-PCR) primer sequences and PCR cycle information are indicated in the Materials and Methods

Increased CCL2 Secretion, but Not EDN1 Secretion, in EC+PBMC Cocultures

Previous studies demonstrated that treatment of bovine luteal EC with the cytokines TNF and IFNG stimulated CCL2 secretion [8]. The present experiments were performed to determine whether or not coculture of EC with PBMC or ActPBMC would stimulate CCL2 secretion. As determined using an ELISA for CCL2, EC and PBMC secreted detectable but modest quantities of CCL2 under basal conditions. CCL2 secretion by EC+PBMC cocultures averaged 5-fold greater than cultures containing either EC or PBMC alone (Fig. 3, experiment I). As expected, EC exposed to recombinant bovine IFNG (positive control) resulted in an 8-fold increase in CCL2 secretion compared with EC alone (Fig. 3).



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  		FIG. 3. Secretion of CCL2 by endothelial cells (EC) of bovine CL (Day 60 of pregnancy) and peripheral blood mononuclear cells (PBMC) over 48 h as determined by immunoassay of conditioned culture medium. Cocultures consisted of EC cultured in direct contact with PBMC (experiment I) and concanavalin A-activated PBMC (ActPBMC; experiment II), or exposed to PBMC- and ActPBMC-conditioned medium (Supernatant; experiments I and II). Bars represent mean protein secretion + SEM (n = 6 experiments). Bars with different letters are significantly different from one another (P < 0.05). There was no effect of PGF2{alpha} within any of the treatment groups and no interaction between coculture conditions and PGF2{alpha} (P > 0.05). Secretion of CCL2 by EC exposed to bovine recombinant IFNG (200 IU/ml) under identical culture conditions is depicted as a positive control

Exposure of EC to concanavalin A increased CCL2 secretion compared with controls (490 ± 51 vs. 85 ± 28 pg/ ml for EC [+Con A] and EC alone, respectively; Fig. 3). In contrast, very low concentrations of CCL2 were detected in media from ActPBMC (Fig. 3, experiment II). However, coculture of EC with ActPMBC increased CCL2 secretion an average of 2-fold greater than concanavalin A-treated EC (Fig. 3, experiment II). The resulting increase in CCL2 was 12-fold higher when compared with controls (Fig. 3, experiment I). The EC+ActPBMC cocultures also produced consistently more CCL2 than EC+PBMC cocultures (1031 ± 177 vs. 300 ± 93 pg/ml, respectively; Fig. 3).

There was no effect of PGF2{alpha} and no interaction between coculture conditions and PGF2{alpha} on CCL2 secretion by EC in either experiments I or II (P > 0.05; Fig. 3). Treatment of EC with PBMC-conditioned medium (supernatants) did not increase secretion of CCL2 (Fig. 3, experiment I). Similarly, treatment of EC with ActPBMC-conditioned medium did not increase secretion of CCL2 compared with concanavalin A-treated EC (Fig. 3, experiment II).

EDN1 secretion by EC+PBMC cocultures did not differ from cultures containing EC alone (P > 0.05; Fig. 4, experiment I) but was greater than that of PBMC alone (Fig. 4, Experiment I). There was no effect of PGF2{alpha}, concanavalin A, or ActPBMC on EDN1 secretion in these experiments (P > 0.05; Fig. 4, experiments I and II). Treatment of EC with IFNG did not stimulate EDN1 secretion (Fig. 4, Experiment I).



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  		FIG. 4. Secretion of EDN1 by endothelial cells (EC) of bovine CL (Day 60 of pregnancy) and peripheral blood mononuclear cells (PBMC) over 48 h as determined by immunoassay of conditioned culture medium. Cocultures consisted of EC cultured in direct contact with PBMC (experiment I) and concanavalin A-activated PBMC (ActPBMC; experiment II), or exposed to PBMC- and ActPBMC-conditioned medium (Supernatant; experiments I and II). Bars represent mean protein secretion + SEM (n = 6 experiments). There were no differences in EDN1 secretion among treatment groups (P > 0.05). There was no effect of PGF2{alpha} within any of the treatment groups and no interaction between coculture conditions and PGF2{alpha} (P > 0.05). EDN1 was not detectable in cultures of PBMC or ActPBMC alone (asterisk). Secretion of EDN1 by EC exposed to bovine recombinant IFNG (200 IU/ml) under identical culture conditions is depicted as a positive control

CCL2 and EDN1 in MLC+PBMC Cocultures

Similar to PBMC, cultures of bovine MLC secreted relatively low concentrations of CCL2. CCL2 secretion by MLC+PBMC cocultures was modest and did not differ from MLC alone or from PBMC alone (P > 0.05; Fig. 5, experiments III and IV). Conversely, CCL2 secretion by MLC+ActPBMC cocultures was greater than MLC alone but did not differ from ActPBMC alone (P > 0.05; Fig. 5, experiments III and IV). There was no effect of PGF2{alpha} (P > 0.05) on CCL2 secretion by MLC cultures, regardless of culture conditions (interaction between coculture and PGF2{alpha}; P > 0.05; Fig. 5). MLC exposed to recombinant bovine IFNG (positive control) secreted 25 times more CCL2 than MLC alone (Fig. 5). The amount of CCL2 secreted by MLC in response to IFNG was comparable with the amount secreted by MLC+ActPBMC cocultures. However, CCL2 secretion by MLC under these stimulatory conditions was much less (approximately one third) than that of EC in the presence of IFNG or ActPBMC (Fig. 3).



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  		FIG. 5. Secretion of CCL2 by mixed luteal cells (MLC) of bovine CL (Day 12 of the estrous cycle) and peripheral blood mononuclear cells (PBMC) over 48 h as determined by immunoassay of conditioned culture medium. Cocultures consisted of MLC cultured in direct contact with PBMC and concanavalin A-activated PBMC (ActPBMC; experiments III and IV). Bars represent mean protein secretion + SEM (n = 3 experiments). Bars with different letters are significantly different from one another (P < 0.05). There was no effect of PGF2{alpha} within any of the treatment groups and no interaction between coculture conditions and PGF2{alpha} (P > 0.05). Secretion of CCL2 by MLC exposed to bovine recombinant IFNG (200 IU/ml) under identical culture conditions is depicted as a positive control

The EDN1 concentration in MLC cultures was below assay detection in all experiments, regardless of culture conditions (results not shown).

Detection of PTGFR in Cell Preparations

PTGFR mRNA was detected in MLC preparations as expected [8]. However, based on Northern blot analysis, no PTGFR was detected in the samples of total RNA derived from PBMC or from EC (Fig. 6).



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  		FIG. 6. Representative Northern analysis of PTGFR mRNA in samples derived from MLC, PBMC, and EC. A 32P-labeled cDNA PTGFR probe was used for hybridization as described in the Materials and Methods section. Fifteen micrograms of each sample were evaluated and the 18S RNA levels served to verify loading. The Northern analysis was repeated three times with different samples. No PTGFR was detected in the samples derived from PBMC or EC

Activated PBMC Inhibit Progesterone Production by Mixed Luteal Cells

Progesterone secretion by MLC increased in response to PGF2{alpha} but was inhibited by coculture with ActPBMC (interaction between PGF2{alpha} and coculture; P < 0.05; Fig. 7). Specifically, coculture of MLC and ActPBMC in the presence of PGF2{alpha} inhibited progesterone secretion compared with controls (Fig. 7). Similarly, progesterone secretion by MLC was decreased following treatment with recombinant bovine IFNG (Fig. 7).



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  		FIG. 7. Progesterone secretion by mixed luteal cells (MLC) of bovine CL (Day 12 of the estrous cycle) cocultured with peripheral blood mononuclear cells (PBMC) over 48 h as determined by radioimmunoassay of conditioned culture medium. Cocultures consisted of MLC cultured in direct contact with PBMC and concanavalin A-activated PBMC (ActPBMC; experiments III and IV). Bars represent mean steroid secretion + SEM (n = 3 experiments). The interaction between PGF2{alpha} and coculture was significant (P < 0.05). Bars with different letters are significantly different from one another (P < 0.05). Secretion of progesterone by MLC exposed to bovine recombinant IFNG (200 IU/ml) under identical culture conditions is depicted as a positive control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Interactions between immune cells and EC of the bovine CL were investigated in the current study to elucidate potential immune-mediated aspects of spontaneous and PGF2{alpha}-induced luteal regression. We determined that endothelial cells were the major source of the proinflammatory mediators CCL2 and EDN1 when compared with cultures of immune cells or luteal cells. Furthermore, our results clearly show that immune cells, specifically PBMC, provoke EC secretion of CCL2 and that this effect is not regulated directly by PGF2{alpha}. In contrast, activated immune cells inhibited progesterone secretion but did not alter the secretion of EDN1. The results provide new evidence that immune cells participate in distinct events associated with corpus luteum regression.

Secretion of CCL2 was highest in cocultures that permitted physical contact between EC of the CL and PBMC and to a lesser extent between steroidogenic cells of the CL (i.e., MLC) and PBMC, especially when activated PBMC were introduced into the coculture. Others have noted similar increases in CCL2 as a result of coculture of immune cells and other epithelial cell types [41–43]. In brain tissue, astrocytes are a source of CCL2 but do not produce detectable amounts of CCL2 in culture unless cocultured with monocytes [41]. Under these conditions, the astrocytes secrete CCL2 in a time-dependent manner that entails physical contact (via adhesion molecules) with the monocytes and involves soluble mediators such as interleukin 1ß and TNF [41]. Similarly, adhesion of macrophages to mesangial cells induces CCL2 expression, and this induction is mediated in part by activation of nuclear factor kappa B [42], a common intracellular signal molecule of cytokine stimulation. The PBMC and EC or MLC cocultures of the current study contained detectable amounts of the cytokine IFNG, but the concentration of IFNG did not differ from cultures of ActPBMC alone. These findings suggest that physical interaction between PBMC and EC of the CL enhances CCL2 expression and that IFNG and/or soluble mediators of activated immune cells may be required to elicit this response. Further studies are needed, however, to clearly determine the relative importance of physical contact (e.g., via adhesion molecules, major histocompatibility complex molecules, etc.) compared with other factors (i.e., soluble mediators such as cytokines, oxygen metabolites, etc.) that might influence interactions between immune cells and other cells within the CL during regression.

In contrast with the increases in CCL2 secretion due to EC-PBMC and MLC-PBMC interactions, these same coculture conditions did not affect EDN1 secretion. The fact that EDN1 in MLC cultures was undetectable indicates that luteal steroidogenic cells express very little, if any, EDN1 in vitro. Conversely, the secretion of EDN1 by cultured EC was high (~2 ng/ml) and was unaffected by coculture with PBMC or treatment with PGF2{alpha}. Here and in a previous study [8], we have shown that EC derived from CL of pregnant cows are devoid of PTGFR. This suggests that the putative effect(s) of PGF2{alpha} on EC and the expression of EDN1 signaling components by EC result from intermediate contributors, most likely cytokines. But as the results of the present study show, activated PBMC (which secreted ~300 pg/ml of IFNG in vitro) and PGF2{alpha} do not affect EC secretion of EDN1. Our findings (current study and [8]) differ from those of Girsh and coworkers [19], who observed a direct effect of PGF2{alpha} on EC secretion of EDN1. It is conceivable that differences in cell culture conditions and/or the phenotypes of the microvascular EC used experimentally may have contributed to the variance in PGF2{alpha} responsiveness. Phenotypic and functional diversity is clearly evident in microvascular EC derived from bovine CL [24]. It is also plausible that the isolation and continued culture of the EC under growth conditions, as implemented in the current study, may have diminished PTGFR expression, dysregulated physiological mechanisms of EDN1 synthesis in response to PGF2{alpha}, and resulted in constitutive EDN1 secretion. Further examination of EDN1 secretion by EC in vitro will be necessary to explore these possibilities.

EDN1 is thought to contribute to luteal regression in several species, including the cow, rat, sheep, and human [10, 20, 44, 45], wherein EDN1 and ET receptor mRNA increase [46]. Furthermore, treatment of animals with PGF2{alpha} at midluteal phase elevates mRNA encoding endothelin converting enzyme (ECE1), ETa, and EDN1 [15]. A subluteolytic dose of PGF2{alpha} coupled with an intramuscular injection of EDN1 in sheep causes a rapid decline in progesterone production and shortens the length of the estrous cycle [45, 47]. Conversely, intraluteal administration of an EDN1 receptor antagonist mitigates the luteolytic effect of PGF2{alpha} and prolongs the luteal phase [45]. Despite evidence for a role in functional regression (inhibition of progesterone), to date, there is no evidence to suggest that EDN1 directly affects the viability of luteal endothelial or steroidogenic cells [24]. Indeed, the high levels of EDN1 present in the current study support the idea that EDN1 does not induce endothelial cell death within the CL. Additional studies evaluating the expression of specific ET receptors and cellular responses to EDN1 will help verify the actions and role of EDN1 on luteal EC.

It is noteworthy that progesterone secretion by MLC was lowest in cultures containing activated PBMC, indicating that a factor(s) of activated PBMC (e.g., IFNG and TNF) or perhaps their contact with luteal steroidogenic cells impairs progesterone production. In contrast, treatment of MLC with PGF2{alpha} served to increase progesterone secretion and cell-cell interaction between luteal steroidogenic cells, and PBMC did not alter progesterone. These findings reinforce assertions by others that immune cell-derived cytokines inhibit luteal progesterone production [48, 49]. The identity of the antisteroidogenic factor or factors secreted by activated immune cells within the CL [50] remains to be determined. Under the present coculture conditions, IFNG appears to meet this requirement because IFNG was secreted by activated PBMC. However, other cytokines may also contribute in conjunction with IFNG to inhibit progesterone secretion [51]. The demonstration that activated PBMC impair luteal progesterone secretion provides an important clue for future studies to identify immune-cell products that regulate steroidogenesis.

Based on current observations of CCL2, EDN1, and progesterone secretion in vitro, luteal steroidogenic cells likely serve a direct role in PGF2{alpha} reception and an intermediate role in the induction of an immune response within the CL during luteal regression. Cultures of MLC, which contain predominantly steroidogenic cells, secreted moderate but variable amounts of CCL2 and undetectable amounts of EDN1 when cocultured with PBMC in the present study. A more robust response in CCL2 secretion occurred following IFNG treatment. This modest, but consistent, response of MLC to IFNG (relative to pure EC) might be attributable to contaminating EC within the MLC cultures. That is, MLC cultures typically contain some EC as a result of the CL dissociation process. Under serum-free conditions, it is estimated that MLC cultures contain less than 5% endothelial cells (Dr. Joy L. Pate, personal communication). In the current study, the MLC cultures were established initially as serum free and then transitioned to medium containing 10% FBS during the last 48 h of coculture with PBMC. Such conditions might promote EC proliferation within the MLC cultures and affect responses to treatment. An intriguing issue raised by these MLC experiments is the possibility that the EC become more responsive to cytokine treatment in the presence of luteal steroidogenic cells. As seen in the current study, for example, CCL2 secretion in MLC cultures increased by 25-fold following IFNG stimulation but only by 8-fold in pure EC cultures. Nevertheless, overall secretion of CCL2 and EDN1 in the MLC cultures was considerably less (5–2000-fold less) than pure cultures of EC, regardless of PGF2{alpha}, PBMC, or cytokine influence. These results and those of previous studies [7, 8, 11] argue strongly in favor of EC as the primary source of CCL2 and EDN1 secretion within the CL, especially considering that the CL is composed predominantly of EC (approximately 50% of the total cell composition [52, 53]). Steroidogenic cells of the CL might play a pivotal role in mediating PGF2{alpha}-induced activation of immune cells, which in turn leads to increased release of proinflammatory molecules (e.g., IFNG, TNF, CCL2, EDN1), promotes an immune response via major histocompatibility complex molecules [32, 50], and facilitates tissue remodeling and cell death during luteal regression [54]. Identifying the mechanism(s) by which luteal steroidogenic cells influence immune cells and possibly EC within the CL is a key element to understanding the luteolytic effect of PGF2{alpha}.

In summary, EC of the bovine CL are a potent source of both CCL2 and EDN1. Coculture of EC with PBMC and activated PBMC augments only the expression of CCL2. In cocultures with MLC, activated PBMC impair progesterone production, an effect attributable to PBMC-derived, soluble mediators (e.g., IFNG) and possibly cell-cell contact but not influenced by PGF2{alpha}. These results reinforce the complex relationship of immune, endothelial, and steroidogenic cell interactions within the CL and raise a number of questions with respect to the mechanism(s) by which PGF2{alpha} acts upon steroidogenic cells directly to then influence endothelial-cell and immune-cell activity during the process of luteal regression.


   ACKNOWLEDGMENTS
 
The authors thank the Tsang and Smith laboratories at the University of New Hampshire for performing the progesterone RIA and providing technical assistance with the RT-PCR procedures.


   FOOTNOTES
 
1 This is scientific contribution 2238 from the New Hampshire Agricultural Experiment Station. The work was supported by USDA grant 2002-35203-12257, NIH HD35934, and the VA. Back

2 Correspondence: David Townson, 509 Kendall Hall, Durham, NH 03824-3590 FAX: 603 862 3758; dave.townson{at}unh.edu Back

Received: 9 June 2004.

First decision: 25 June 2004.

Accepted: 12 January 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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