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Endocrinology Vol. 143, No. 9 3582-3589
Copyright © 2002 by The Endocrine Society

ARTICLE

Secretion of Monocyte Chemoattractant Protein-1 by Endothelial Cells of the Bovine Corpus Luteum: Regulation by Cytokines But Not Prostaglandin F2{alpha}

Victoria A. Cavicchio, James K. Pru, Benjamin S. Davis, John S. Davis, Bo R. Rueda and David H. Townson

Department of Animal and Nutritional Sciences (V.A.C., D.H.T.), University of New Hampshire-Durham, Durham, New Hampshire 03824; Vincent Center for Reproductive Biology (B.R.R., J.K.P.), Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114; Women’s Research Institute (B.S.D., J.S.D.), University of Kansas School of Medicine-Wichita, Veterans Affairs Medical Center, Wichita, Kansas 67214; and Olson Center for Women’s Health (J.S.D.), Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, Nebraska 68198

Address all correspondence and requests for reprints to: David H. Townson, Ph.D., Department of Animal and Nutritional Sciences, University of New Hampshire, 128 Main Street, Durham, New Hampshire 03824. E-mail: dave.townson{at}unh.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Information regarding the regulation of monocyte chemoattractant protein-1 (MCP-1) in regression of the corpus luteum (CL) is limited. This study tested the hypothesis that endothelial cells derived from bovine CL are a source of MCP-1, and that proinflammatory cytokines, prostaglandin F2{alpha} (PGF2{alpha}), and progesterone regulate MCP-1 expression. Endothelial cells were treated without (Control) or with PGF2{alpha} (1 µM), TNF{alpha} (100 ng/ml), interferon-{gamma} (IFN{gamma}, 200 IU/ml), and TNF{alpha} + IFN{gamma} for 24 and 48 h in the absence or presence of progesterone (P4, 250 ng/ml). Increases in MCP-1 mRNA and protein were observed in response to TNF{alpha} within 24 and 48 h of culture, respectively (P < 0.05). Interferon-{gamma} stimulated (P < 0.05) both MCP-1 mRNA and protein after 24 h of culture, and this effect was also sustained through 48 h of culture (P < 0.05). Cotreatment of cultures with TNF{alpha} + IFN{gamma} lead to further increases (P < 0.05) in MCP-1 in both 24- and 48-h cultures. Surprisingly, neither PGF2{alpha} nor P4 affected MCP-1 production. Subsequent experiments revealed that the endothelial cells lacked prostaglandin F2{alpha} receptor mRNA, and the MAPK pathway, although present and responsive to growth factor stimulation, was unresponsive to PGF2{alpha} stimulation. In summary, endothelial cells derived from bovine CL respond to TNF{alpha} and IFN{gamma} stimulation with an increase in MCP-1 secretion. In contrast, neither PGF2{alpha} nor P4 directly influenced endothelial expression of MCP-1. These results suggest that cytokines stimulate the synthesis of MCP-1 observed during PGF2{alpha}-induced luteal regression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CORPUS LUTEUM (CL) is a transient endocrine tissue that forms within the ovary from the remnants of the ovulated follicle and is responsible for the regulation of the estrous cycle and the maintenance of pregnancy through the production of progesterone (P4). The CL is comprised primarily of steroidogenic cells, endothelial cells, and fibroblasts (1, 2, 3, 4). Immune cells such as macrophages, eosinophils, and T-lymphocytes also reside within the tissue and are thought to influence luteal function and/or integrity (5, 6, 7, 8, 9). In domestic farm animals, the process by which the CL undergoes spontaneous degeneration, luteolysis, is initiated by the release of uterine prostaglandin F2{alpha} (PGF2{alpha}) if conception does not occur (10, 11). However, the sustained involvement of PGF2{alpha} in the decline of steroidogenesis and the degeneration of the CL during regression is less certain. Other mediators of steroidogenesis, luteal cell fate, and immune response mechanisms may be involved, and in recent years have received an increased level of attention.

Results from recent studies indicate that the chemokine, monocyte chemoattractant protein-1 (MCP-1), is produced in the CL during luteal regression and might aid in its destruction (7, 9, 12, 13, 14, 15, 16). MCP-1 is of particular interest because, once expressed within blood vessels, this chemokine facilitates the attachment and migration of immune cells, specifically monocytes, macrophages, and T-lymphocytes, from the blood stream into sites of inflammation (17). In this regard, macrophages and T-lymphocytes have been demonstrated to accumulate in regressing CL of many species (8, 9, 12, 15, 18, 19) and have been implicated in phagocytosis of luteal cells (20), degradation of extracellular matrix (21), and secretion of proinflammatory mediators that influence luteal steroidogenesis (22, 23).

Although it is appreciated that MCP-1 expression increases during luteal regression, little is known about the cellular source(s) and factors that regulate MCP-1 expression in the ovary. In recent studies, the endothelial cells of the bovine CL have been identified as a putative source of MCP-1 secretion (9, 15). The current study extends these observations and examines the potential for direct regulation of MCP-1 secretion by proinflammatory mediators associated with luteal regression. Specifically, we tested the hypothesis that endothelial cells derived from bovine CL are a source of MCP-1, and that proinflammatory cytokines, PGF2{alpha}, and P4 directly regulate mRNA expression and protein secretion of MCP-1. In this report, we present data demonstrating that TNF{alpha} and interferon-{gamma} (IFN{gamma}), but not PGF2{alpha} or P4, elevate MCP-1 secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
All antibodies, enzymes, and kits were used according to the manufacturer’s specifications. Endothelial cell growth medium and basal medium (EBM-2) were obtained from Clonetics (San Diego, CA). Plastic tissue culture vessels, 6-, 12-, and 24-well plates, and T75 flasks were obtained from Corning, Inc. (Corning, NY). Enhanced chemiluminescence kits were purchased from Amersham (Arlington Heights, IL), Immobilon-p [polyvinylidene difluoride (PVDF)] membranes were purchased from Millipore Corp. (Bedford, MA), [{alpha}-32P]deoxy-CTP and Hyperfilm were obtained from Amersham Pharmacia Biotech (Piscataway, NJ) and 18S rRNA from Ambion, Inc. (Austin, TX). DuoSet hMCP-1 sandwich ELISA development system was obtained from R & D Systems (Minneapolis, MN). Protease and phosphatase I and II inhibitor cocktails, PGF2{alpha}, P4, TriReagent and prestained molecular weight markers were purchased from Sigma (St. Louis, MO). Bovine MCP-1 cDNA was generously donated by Dr. Hellmut Augustin (Institute of Molecular Oncology, Tumor Biology Center, Freiburg, Germany). Bovine prostaglandin F2{alpha} receptor (FP) cDNA was synthesized using primer sequences published previously by Mamluk et al. (24). Murine TNF{alpha} was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY), and bovine IFN{gamma} was a generous gift of Dr. Dale Godson (Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada). Human IGF-I (hIGF-I) was obtained from Life Technologies, Inc. (Grand Island, NY) and murine epidermal growth factor (mEGF) was from Sigma. Phosphorylation state-specific antibodies for the activated forms of ERK-1 and ERK-2 (p44 and p42 MAPK, respectively) and for pan-ERK (nonphosphorylated, monophosphorylated, and dually phosphorylated forms of ERK-1 and ERK-2) were obtained from Promega Corp. (Madison, WI). All other chemicals and reagents were purchased from Sigma or Fisher Scientific (Pittsburgh, PA).

Cell isolation and culture
Purified endothelial cell populations were purchased from Cambrex Biosciences (BioWhittaker, Inc., Walkersville, MD). Briefly, CL were obtained from cows in early pregnancy at a local slaughterhouse (Quality Meats, Wellington, KS) and transported to the laboratory in ice-cold M199 containing 0.1% BSA. The CL were rinsed with fresh sterile EBM-2 and shipped overnight to Cambrex Biosciences in EBM-2 on wet ice. The CL were sliced and endothelial cells released by enzymatic dissociation procedures. Purity of the resultant endothelial cells was more than 99% as determined by immunostaining for von Willebrand factor and by uptake of fluorescent labeled acetylated low density lipoprotein (Ac-LDL).

In the present study, endothelial cells from frozen aliquots (passage 2) were plated in 6-, 12-, and 24-well plates and in T75 flasks (seeding density 5000–7500 cells/cm2). The cells were cultured in endothelial cell growth medium, as provided by the supplier except that the fetal bovine serum concentration was used at 3%, and P4 (250 ng/ml) was added to approximate progesterone levels in the CL. The cells were cultured at 37 C in a humidified chamber of 5% CO2 in air for 6–7 d with medium exchanges every 48 h. Under these conditions, the cultures reached approximately 90% confluency. Before experiments, the medium was changed and the cultures were maintained thereafter in EBM-2 for 24 h without or with P4 (250 ng/ml). Medium was then removed and fresh medium was replaced for an equilibration period of 3 h before treatment administration. Treatments consisted of endothelial cells exposed to vehicle (Control), TNF{alpha} (100 ng/ml), IFN{gamma} (200 IU/ml), TNF{alpha}+ IFN{gamma}, PGF2{alpha} (1 µM) for 24 or 48 h. Treatment concentrations were based upon previously published in vitro studies of mixed luteal cells (25, 26, 27).

Endothelial cell uptake of Ac-LDL fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI)-labeled probe
To confirm the purity of the cells obtained from Cambrex Biosciences, the endothelial cells were cultured as described above and then serum starved for 24 h before incubation with Ac-LDL conjugated with DiI. DiI-Ac-LDL uptake is an established criterion for endothelial cell characterization (28). Bovine pulmonary aortic endothelial cells obtained from American Type Culture Collection (catalog no. CCL 209) were cultured as a positive control.

MCP-1 ELISA
The secretion of MCP-1 into culture medium was measured using a commercially available sandwich ELISA kit (Duo-set, R&D Systems, Minneapolis, MN). The assays were run in 96-well plates using 100 µl of medium according to the manufacturer’s instructions. Use of this assay kit for the detection of bovine MCP-1 was previously validated by others (29). For standards, human recombinant MCP-1 was used at concentrations ranging from 15.6–1000 pg/ml. The detection limit of the assay was 10.5 pg/ml; and mean interassay and intraassay coefficients of variation were 10.3% and 4.2%, respectively. Results were expressed as picograms of MCP-1 per milliliter of conditioned medium.

Measurement of MCP-1 and FP mRNA
Total cellular RNA was isolated from cultured endothelial cells using TriReagent in accordance with the manufacturer’s instructions. Following isolation, total RNA (10 µg) was separated by 1.5% agarose:formaldehyde gel electrophoresis at 100 V for 2.5 h. The total RNA was then transferred to nylon membranes (0.2 µm) by capillary blotting for 15–18 h. RNA was fixed to membranes (2 h, 80 C) and prehybridized for 3 h at 42 C as previously described (30). The blots were hybridized overnight at 42 C with the appropriate cDNA probe (bMCP-1 or bFP, 18S rRNA) primed with 50 µCi [{alpha}-32P]deoxy-CTP. Blots were washed as described previously (30) and exposed to film. The blots were stripped (0.1% sodium dodecyl sulfate) and reprobed to detect 18S rRNA for the purpose of normalization. Scanning and densitometric analyses were performed using a Kodak Image Analysis System (Eastman Kodak Co., Rochester, NY).

Western blots for phosphorylated ERK-1 and ERK-2 proteins
Endothelial cells were treated with PGF2{alpha} (1 µM), hIGF-I (50 ng/ml), or mEGF (5 ng/ml) for 10 min at 37 C. Experiments were terminated by rinsing the cells with ice-cold PBS. The cells were lysed with cold lysis buffer (10 mM Tris-HCl; 1 mM EDTA; 1 mM EGTA; 100 mM NaCl; 1% Triton X-100; 0.5% Nonidet P-40, pH 7.4) containing protease and phosphatase inhibitor cocktails and kept on ice for 10 min. The cells were scraped from wells and further lysed by passage through a 28-gauge needle, then collected and stored at -80 C for analysis.

Proteins obtained from endothelial cell lysates (~13 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes as previously described (23, 31). Western blot analysis was performed using an antibody that recognizes the active (phosphorylated) forms of ERK-1 and -2. Subsequently, the membranes were stripped and reprobed with pan-ERK to verify equal loading and normalization as described previously (23, 26, 27).

Statistics
All of the data were subjected to one-way ANOVA using the general linear model procedure of Minitab (State College, PA); a Tukey’s test for pairwise comparisons was used to further compare means among treatment groups. Results are expressed as the mean ± SEM unless otherwise indicated. Each experiment was repeated a minimum of three times, using a separate preparation of isolated endothelial cells for a given experiment. Figure legends indicate the number of times an experiment was repeated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Endothelial cell uptake of Ac-LDL fluorescent DiI-labeled probe
The endothelial cells grew into confluent monolayers, their growth was density inhibited, and they exhibited an elongate, spindle-shaped morphology typical of endothelial cells (Fig. 1Go). The morphology of the cells under phase contrast (Fig. 1AGo) and under fluorescence microscopy following DiI-Ac-LDL uptake (Fig. 1BGo) was consistent with a pure population of endothelial cells (>99%).



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  		Figure 1. Endothelial cell uptake of DiI-Ac-LDL. Phase contrast (x37.5 magnification, automatic exposure) and fluorescent (x37.5, 2-sec exposure) views (A and B, respectively) of confluent endothelial cells in culture depicting characteristic endothelial cell morphology, and uptake of Ac-LDL conjugated with DiI. Cytoplasmic fluorescent red staining indicates DiI-Ac-LDL uptake.

 
Cytokines stimulate MCP-1 mRNA in luteal-derived endothelial cells
Northern blot analysis indicated an induction of MCP-1 mRNA in the endothelial cell cultures following treatment with TNF{alpha}, IFN{gamma}, and TNF{alpha} + IFN{gamma} (Fig. 2AGo). A significant increase in MCP-1 mRNA was observed after 24 h of culture in the presence of TNF{alpha} and/or IFN{gamma} (P < 0.05; Fig. 2BGo). Treatment with PGF2{alpha}, however, did not affect MCP-1 mRNA (P > 0.05; Fig. 2Go, A and B). Similarly, there was no effect of P4 on basal or cytokine stimulated MCP-1 mRNA (results not shown).



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  		Figure 2. MCP-1 mRNA in endothelial cells. A, Representative Northern blot showing MCP-1 mRNA expression in endothelial cells untreated (Control) or treated with TNF{alpha} (100 ng/ml), IFN{gamma} (200 IU/ml), TNF{alpha} + IFN{gamma}, or PGF2{alpha} (1 µM) for 24 h. In the lower panel, 18S rRNA is depicted to show consistent loading. B, Quantitation of MCP-1 mRNA (x103 pixels) in response to 24 h cytokine treatment (mean ± SEM). Different letters denote differences in MCP-1 mRNA between treatment groups (P < 0.05). This experiment was repeated three times with similar results.

 
Cytokines stimulate MCP-1 secretion by luteal-derived endothelial cells
Similar to the changes noted above in MCP-1 mRNA, TNF{alpha} and IFN{gamma} stimulated MCP-1 secretion by endothelial cells (Fig. 3Go). Basal secretion of MCP-1 was evident in conditioned medium of control cultures and did not change over time (P > 0.05). In contrast, TNF{alpha} stimulated MCP-1 secretion more than 2-fold by 24 h (P < 0.05), and more than 7-fold by 48 h compared with controls (P < 0.05; Fig. 3Go). Treatment with IFN{gamma} stimulated MCP-1 secretion 4- and 9-fold above controls (P < 0.05, 24 and 48 h of culture, respectively; Fig. 3Go). Cotreatment with TNF{alpha} + IFN{gamma} further stimulated MCP-1 secretion (P < 0.05; 7- and 16-fold above controls at 24 and 48 h, respectively; Fig. 3Go). Treatment with PGF2{alpha} or P4 had no effect on MCP-1 secretion in these experiments (results not shown).



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  		Figure 3. Secretion of MCP-1 by cultured endothelial cells. Endothelial cells were left untreated (Control) or treated with TNF{alpha} (100 ng/ml), IFN{gamma} (200 IU/ml), TNF{alpha} + IFN{gamma}, or PGF2{alpha} (1 µM) for 24 or 48 h. Bars represent mean protein secretion ± SEM as detected by ELISA (sensitivity 10.5 pg/ml). Different letters denote differences in MCP-1 protein secretion between treatment groups (P < 0.05). An apostrophe denotes differences between 24 h and 48 h of culture within treatment group (P < 0.05). This experiment was repeated seven times with similar results.

 
PGF2a fails to stimulate phosphorylation of ERK isoforms in luteal-derived endothelial cells
One of the early responses to PGF2{alpha} involves the activation of the ERK MAPK pathway and the expression of the early response gene c-fos (26, 27). Phosphorylation of ERK-1 and ERK-2 was determined by Western blotting to verify the existence of an active MAPK pathway within the endothelial cells and to assess the sensitivity of the MAPK pathway to PGF2{alpha} stimulation. Treatment with hIGF-I and mEGF provoked significant increases in the phosphorylation of both ERK-1 and ERK-2 (P < 0.05), indicative of MAPK activity, whereas PGF2{alpha} failed to elicit a similar response (P > 0.05; Fig. 4Go, A and B).



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  		Figure 4. Activation of the MAPK pathway by cultured endothelial cells. A, Representative Western blot depicting phospho-ERK-1 and phospho-ERK-2 in endothelial cells following treatment without (Control) or with PGF2{alpha} (1 µM), hIGF-I (50 ng/ml) or mEGF (5 ng/ml). In the lower panel, pan-ERK, recognizing both phosphorylated and nonphosphorylated ERK isoforms, is depicted to show consistent loading. B, Quantitation of phospho-ERK levels in protein lysates from endothelial cell cultures following 10 min treatment with PGF2{alpha}, hIGF-I or mEGF. Different letters denote differences in ERK phosphorylation between treatment groups (P < 0.05). This experiment was repeated three times with similar results.

 
Luteal-derived endothelial cells lack receptors for PGF2{alpha} (FP)
The lack of responsiveness to PGF2{alpha} treatment suggested a problem with PGF2{alpha} reception. Northern analysis indicated a lack of FP mRNA in endothelial cells following treatment with control media, TNF{alpha}, IFN{gamma}, TNF{alpha} + IFN{gamma}, and PGF2{alpha} for 24 h (Fig. 5Go, lanes 1–5). In contrast, abundant FP mRNA was evident in corpora lutea obtained on d 6, 12, and 18 of the estrous cycle (lanes 6–8). The FP transcripts migrated to the appropriate sizes as previously reported (32).



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  		Figure 5. FP mRNA in endothelial cells and bovine CL. Northern blot depicting FP mRNA expression in cultured endothelial cells and in luteal tissue from bovine CL. FP mRNA was not detected in endothelial cells untreated (Control) or treated with TNF{alpha} (100 ng/ml), IFN{gamma} (200 IU/ml), TNF{alpha} + IFN{gamma}, or PGF2{alpha} (1 µM) for 24 h (lanes 1–5). In contrast, FP mRNA was detected in CL from d 6, 12, and 18 post ovulation (lanes 6–8).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study examined the direct effects of proinflammatory cytokines, PGF2{alpha}, and P4 on MCP-1 expression by endothelial cells derived from the bovine CL. Our findings indicate that luteal-derived endothelial cells are clearly a source of MCP-1 and respond directly to cytokine (i.e. TNF{alpha} and IFN{gamma}) stimulation. Increases in both mRNA levels and protein secretion of MCP-1 were evident in the endothelial cell cultures following cytokine treatment, but the cultures did not respond whatsoever to treatment with either PGF2{alpha} or P4. The lack of a direct effect of PGF2{alpha} on MCP-1 expression in vitro was unexpected, and may be significant considering that previous in vivo studies have shown an increase in MCP-1 mRNA expression during PGF2{alpha}-induced regression of the CL (7, 14, 33). Until now, no studies have determined with certainty the cellular sources of MCP-1 within the CL. Tsai et al. (14) and Haworth et al. (33) reported that large steroidogenic cells of the CL do not express MCP-1 mRNA, although these cells are known to possess PGF2{alpha} receptors. Additionally, primary cultures of luteal steroidogenic cells do not respond to PGF2{alpha} with an elevation in MCP-1 levels (34). Penny and co-workers (7) demonstrated that T lymphocytes are a plausible source of MCP-1 mRNA, but these authors readily acknowledged that other, as yet uncharacterized, cell types of the CL might also contribute to MCP-1 secretion. More recently, endothelial cells have been identified as a source of MCP-1 within the bovine CL based upon immunohistochemical evidence (9, 15). Indeed, the present studies demonstrate that luteal-derived microvascular endothelial cells produce substantial quantities of MCP-1 in response to TNF{alpha} and IFN{gamma}, but PGF2{alpha} fails to elicit a similar response. Taken together, these studies lead us to suggest that the ability of PGF2{alpha} to induce luteal MCP-1 expression in vivo is indirect and is perhaps mediated by a variety of cell types and/or cellular factors.

The observation that endothelial cells are a source of MCP-1 in the CL is consistent with previous reports of endothelial-derived MCP-1 expression in other tissues. In human umbilical endothelial cells, MCP-1 mRNA and protein are enhanced in response to proteinase-3, an autoantigen associated with the autoimmune disease, Wegener’s granulomatosis (35). Both human and bovine aortic endothelial cells express MCP-1 mRNA and protein in vitro (29, 36), and endothelial cell fractions of rat liver cells express MCP-1 mRNA after a single exposure to hepatotoxin (37). Interestingly, basal or constitutive expression of MCP-1 was evident in the endothelial cell cultures of the current study; a finding that helps explain the observation of immunodetectable MCP-1 within the vasculature of the bovine CL as early as d 6 post ovulation (9). Further stimulation of MCP-1 secretion in the current study required the presence of TNF{alpha} and/or IFN{gamma}, whereas PGF2{alpha} had no effect. This suggests that the gene encoding MCP-1 within endothelial cells of the bovine CL is induced directly by cytokines, but not by PGF2{alpha}.

The failure of PGF2{alpha} to induce MCP-1 in endothelial cell cultures in the current study led us to question the responsiveness of these cells to PGF2{alpha}. PGF2{alpha} is known to activate the Raf-1/MEK-1/ERK-1/2 phosphorylation signaling cascade in luteal cell cultures from collagenase-dispersed CL that contain steroidogenic cells, and contaminating endothelial cells and fibroblasts (26, 27). This signaling mechanism results in the translocation of PGF2{alpha}-activated ERK-1 and -2 to the nucleus and the phosphorylation of transcription factors that regulate the expression of the early response gene c-fos. Coincidentally, Ishikawa et al. (38) have shown recently that a similar signaling pathway induces MCP-1 mRNA in rat glomeruli. Our results indicate that an active ERK signaling pathway exists in the endothelial cells derived from the bovine CL, but the cells are unresponsive to PGF2{alpha}. The lack of effect on MCP-1 mRNA and protein secretion, in combination with the lack of response by downstream mediators of FP, suggests that these cells may be deficient in receptors for PGF2{alpha} and therefore incapable of direct activation by PGF2{alpha}. In fact, subsequent experiments revealed no evidence of mRNA encoding FP in these cells despite the obvious presence of the message for these receptors in bovine luteal tissue obtained on d 6, 12, and 18 of the estrous cycle. It is possible that the endothelial cells used in this study following one passage may have lost their capacity for PGF2{alpha} responsiveness. However, this seems unlikely because we have found that the response of endothelial cell cultures to cytokine and PGF2{alpha} treatment is similar to the response of primary cultures of mixed luteal cells (i.e. containing both endothelial cells and steroidogenic cells). That is, TNF{alpha} and IFN{gamma} profoundly stimulated MCP-1 expression in mixed luteal cell cultures, whereas PGF{alpha} had no effect (34). These results suggest that the absence of PGF2{alpha} responsiveness and the inability of PGF2{alpha} to induce MCP-1 expression in the current study is not a consequence of cell passage in these cultures of pure endothelial cells. Our results contrast, however, with observations made by Mamluk and colleagues (24) who reported that luteal endothelial cells express FP mRNA, and Girsh et al. (39) who reported that luteal endothelial cells respond to PGF2{alpha} treatment with an increase in endothelin-1 expression. The discrepancy between the present study and the earlier two studies may be attributed to differences in cell culture conditions, the types of microvascular endothelial cells used, or the techniques used to detect FP mRNA. Mamluk et al. (24) and Girsh et al. (39) used type III clonal microvascular endothelial cells (passages 4–10) derived from bovine CL of the estrous cycle (40, 41). These cells had a cobblestone appearance and were cultured on 1% collagen type I (Vitrogen 100). The present study used microvascular endothelial cells (passage 2) derived from bovine CL of pregnancy that had an elongate, spindle-shaped appearance and were cultured on plastic. Others have noted the phenotypic and functional diversity of microvascular endothelial cells derived from the bovine CL (40, 41, 42, 43, 44). Of particular interest are the recent observations by Aust et al. (42), who reported that cytokeratin 18 positive (CK+) endothelial cells from the bovine CL possess functional receptors for PGF2{alpha}, whereas cytokeratin 18 negative (CK-) endothelial cells do not. The CK+ cells are considered to be a rare microvascular endothelial cell within the bovine CL that have a cobblestone appearance and characteristics similar to endothelial cells from the bovine aorta (40, 41, 42, 43, 44). In contrast, CK- cells, the common microvascular endothelial cell found within the bovine CL (40, 41, 42, 43, 44), often exhibit a spindle-shaped morphology, and are described by Lehman et al. (43) as more potent than CK+ microvascular endothelial cells in producing cytokines that are involved in eosinophil and monocyte/macrophage extravasation into the bovine corpus luteum and the maturation/activation of these leukocytes. Thus, a disparity may exist between subpopulations of microvascular endothelial cells within the bovine CL and the ability of these cells to respond to PGF2{alpha}. In addition, the present study used Northern analysis to detect the presence of FP mRNA and produced findings similar to those of others (45); that is, FP transcripts were localized predominantly to steroidogenic luteal cells and not to other compartments of the ovary. Admittedly, the RT-PCR technique reported by Mamluk et al. (24) has much greater sensitivity than Northern blot analysis and may have amplified low copy FP transcripts present in either the endothelial cells or contaminating cells. Despite these apparent differences, the current study demonstrated that the absence of FP mRNA in purified luteal endothelial cells was coupled with a lack of functional responses to PGF2{alpha} (signal transduction and gene expression). This strongly suggests that endothelial cells derived from CL of pregnancy do not respond to PGF2{alpha} because they lack sufficient PGF2{alpha} receptors. Further studies are required to fully characterize the functional differences in FP expression in endothelial cells derived from the CL of the estrous cycle and of pregnancy. However, acknowledging that changes in gene expression may occur in endothelial cells as a result of pregnancy, the absence of FP transcripts in luteal endothelial cells during early pregnancy may be an adaptive mechanism to maintain progesterone production by the CL and thereby maintain a suitable environment for the developing fetus.

Similar to the lack of response to PGF2{alpha}, treatment with P4 did not affect MCP-1 protein secretion, nor did it alter the stimulatory actions of TNF{alpha} and IFN{gamma} on MCP-1 expression. The present results demonstrate that endothelial cells derived from the bovine CL are capable of expressing MCP-1 independent of interactions with steroidogenic luteal cells. In a recent study investigating the effect of P4 administration on MCP-1 expression by eosinophils in the ovine uterus, Asselin et al. (46) showed a significant increase in eosinophil infiltration following P4 treatment. It seems possible, therefore, that P4 may regulate luteal MCP-1 mRNA expression and protein secretion indirectly through the actions of infiltrating immune cells. Luteal endothelial cells are probable targets of a variety of factors produced by these cells (e.g. cytokines, reactive oxygen species, phospholipase A2). Further experiments are necessary to test this idea.

In summary, our results indicate that endothelial cells are an important source of MCP-1 secretion within the bovine CL. The cytokines TNF{alpha} and IFN{gamma} increase endothelial cell production of MCP-1 directly and significantly, whereas PGF2{alpha} and P4 have no effect. These observations suggest that cytokines mediate the increase in MCP-1 within the bovine corpus luteum during PGF2{alpha}-induced luteal regression. As a final note, we and others (24, 40, 41, 42, 43, 44), have provided initial evidence to suggest that the bovine CL contains subpopulations of microvascular endothelial cells, each responding differently to luteolytic agents such as PGF2{alpha} and proinflammatory cytokines.


   Acknowledgments
 
Appreciation is extended to Drs. Dennis Bobilya and Thomas Foxall, Barb Brothwell, and Adele Marone (Department of Animal and Nutritional Sciences, UNH, Durham, NH) for their assistance with the DiI-Ac-LDL experiments and the fluorescent microscopy.


   Footnotes
 
This manuscript is scientific contribution number 2113 from the New Hampshire Agricultural Experiment Station. The work was supported in part by USDA Grant 97-35208-4705 (to D.H.T.), NIH Grant R01-HD-35934 (to B.R.R. and J.S.D.), Vincent Memorial Research Funds (to B.R.R.) and The Department of Veterans Affairs (to J.S.D.). J.K.P. is a Postdoctoral Research Fellow supported by the Lalor Foundation.

Abbreviations: Ac-LDL, Acetylated low density lipoprotein; CL, corpus luteum; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate; EBM-2, endothelial cell basal medium; FP, prostaglandin F2{alpha} receptor; hIGF-I, human IGF-I; IFN{gamma}, interferon-{gamma}; MCP-1, monocyte chemoattractant protein-1; mEGF, murine epidermal growth factor; P4, progesterone; PGF2{alpha}, prostaglandin F2{alpha}.

Received April 9, 2002.

Accepted for publication May 2, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rodgers RJ, O’Shea JD, Bruce NW 1984 Morphometric analysis of the cellular composition of the ovine corpus luteum. J Anat 138:757–769
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