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BOR - Papers in Press, published online ahead of print April 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017590
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BIOLOGY OF REPRODUCTION 69, 695–700 (2003)
DOI: 10.1095/biolreprod.103.017590
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Effects of Prostaglandin F2{alpha} and Progesterone on the Ability of Bovine Luteal Cells to Stimulate T Lymphocyte Proliferation1

Matthew J. Cannon, Margaret G. Petroff3, and Joy L. Pate2

Department of Animal Sciences, The Ohio State University/Ohio Agricultural Research and Development Center, Wooster, Ohio 44691


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bovine luteal cells express class I and II major histocompatibility complex molecules and stimulate T lymphocyte proliferation in vitro. Proliferation of T lymphocytes is greater in cocultures of luteal cells and T lymphocytes collected following administration of a luteolytic dose of prostaglandin (PG) F2{alpha} to the cow. Whether this results from changes in luteal cells that increase their ability to stimulate T lymphocyte proliferation or from changes in T lymphocytes that enhance their ability to respond to luteal cells is unclear. To determine which is the case, luteal cell-T lymphocyte cocultures were performed using luteal cells and T lymphocytes isolated from the same animals before and 8 h after administration of PGF2{alpha}. In the presence of T lymphocytes collected before PGF2{alpha} administration, luteal cells isolated after PGF2{alpha} were more potent stimulators of T lymphocyte proliferation than were luteal cells collected before PGF2{alpha} (P < 0.05). The effect of progesterone on luteal cell-stimulated T lymphocyte proliferation was also evaluated. Proliferation of T lymphocytes was greater (P < 0.05) in cultures containing the cytochrome P450 side-chain cleavage enzyme-inhibitor aminoglutethimide. Exogenous progesterone caused a dose-dependent inhibition of luteal cell-stimulated T lymphocyte proliferation (P < 0.05). Progesterone-receptor mRNA was undetectable in peripheral blood mononuclear cells collected before and after PGF2{alpha} administration, indicating that the effect of progesterone was not mediated via progesterone receptors in lymphocytes. These results imply that specific changes in luteal cells in response to PGF2{alpha} enhance the ability of these cells to stimulate T lymphocyte proliferation. These results also demonstrate that progesterone can suppress luteal cell-stimulated T lymphocyte proliferation.

corpus luteum, immunology, ovary, progesterone, progesterone receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regression of the bovine corpus luteum (CL) is an event that may be mediated in part by the immune system. The proinflammatory cytokines tumor necrosis factor {alpha}, interleukin-1ß, and interferon-{gamma} (IFN-{gamma}) all inhibit LH-stimulated progesterone production and stimulate prostaglandin (PG) synthesis by cultured bovine luteal cells [1–5]. Messenger RNA for these cytokines is present within bovine luteal tissue [6, 7], as are T lymphocytes and macrophages [6, 8]. The presence of class II major histocompatibility complex (MHC) molecules on the cell surface, which provide the means for cells to stimulate activation of CD4+ T lymphocytes, can be induced on bovine luteal cells in vitro by IFN-{gamma} treatment [9]; class II MHC molecules also are present on the bovine luteal cells in vivo [10]. The percentage of luteal cells expressing class II MHC molecules increases late in the estrous cycle, near the time of luteal regression, and also in response to administration of a luteolytic dose of PGF2{alpha} to the cow [10]. When luteal cells from nonpregnant cows at Day 18 postestrus were compared with luteal cells from cows in which an embryo was present in the uterus at Day 18, the percentage of luteal cells expressing class II MHC molecules was greater in nonpregnant animals [10]. Collectively, it can be inferred from these observations that class II MHC molecules play a role in facilitating the process of luteal regression.

Bovine luteal cells are potent stimulators of T lymphocyte proliferation in vitro [11]. Consistent with the hypothesis that MHC molecules on luteal cells are involved in facilitating luteal regression, Petroff et al. [11] demonstrated that luteal cells isolated from regressing CL are more potent stimulators of T lymphocyte proliferation than are luteal cells from fully functional, midcycle CL [11]. Because luteal regression was induced by administration of a luteolytic dose of PGF2{alpha} in that study, it was unclear whether the increase in luteal cell-stimulated T lymphocyte proliferation resulted from effects of PGF2{alpha} on the luteal cells or on T lymphocytes. One possibility is that the ability of luteal cells to stimulate T lymphocyte proliferation was enhanced in response to administration of a luteolytic dose of PGF2{alpha} to the cow. Equally valid is the suggestion that the ability of T lymphocytes to respond to luteal cells increased in response to PGF2{alpha}. Therefore, the present study was conducted to address the hypothesis that luteal cells (rather than T lymphocytes) are altered in response to in vivo administration of PGF2{alpha}, enhancing their ability to stimulate T lymphocyte proliferation.

The objective of the present study was to determine whether the increase in T lymphocyte proliferation observed in cocultures of luteal cells and T lymphocytes collected after in vivo administration of PGF2{alpha} results from changes in luteal cells or, alternatively, in T lymphocytes. Results obtained in the first part of the present study indicated that a decrease in the capacity of luteal cells to synthesize progesterone could be partially responsible for enhancing T lymphocyte proliferation in cocultures of T lymphocytes and luteal cells collected after administration of PGF2{alpha}. Therefore, a second objective of the present study was to evaluate the effects of progesterone on luteal cell-stimulated T lymphocyte proliferation.


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

Culture medium RPMI 1640, powdered Ham F-12 medium, fetal bovine serum, penicillin-streptomycin, glutamine, TRIzol Reagent, and Superscript II RNase H- reverse transcriptase were purchased from Life Technologies (Grand Island, NY). TaqBead Hot Start Polymerase was purchased from Promega (Madison, WI). Random hexamer primers and Ficoll-Paque Plus were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Type I collagenase was acquired from Worthington Biochemical (Freehold, NJ). Anti-bovine immunoglobulin (Ig) G was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Cellect bovine T lymphocyte immunocolumns were acquired from Biotex Laboratories (Edmonton, AB, Canada). Mitomycin C and staphylococcal enterotoxin B were purchased from Sigma Chemical Co. (St. Louis, MO). Ninety-six-well culture plates were purchased from Corning (Corning, NY). All other chemicals were acquired from Sigma or Fisher Scientific (Fairlawn, NJ).

Collection and Dissociation of Luteal Tissue

Normally cycling, multiparous, lactating Holstein and Jersey cows between 3 and 6 yr of age were used in the present study. Animals were housed indoors, had complete freedom of movement, and were fed a total mixed ration ad libitum. Experimental procedures were performed during the midluteal phase (Days 9–12) of the estrous cycle (Day of estrus = Day 0). Twenty-five milligrams of PGF2{alpha} (Lutalyse; Upjohn Co., Kalamazoo, MI) were administered i.m. to cows in the midluteal phase of the estrous cycle to induce luteal regression [12]. Luteal tissue and jugular venous blood were collected from cows before and 8 h after administration of a luteolytic dose of PGF2{alpha}. Before surgical procedures, blood was collected for subsequent determination of plasma progesterone concentrations via ELISA, as described previously [11]. A small portion of the CL, weighing 100–200 mg, was removed transvaginally using a Jackson equine uterine biopsy forceps (Jorgensen Laboratories, Inc., Loveland, CO). Immediately following removal of the luteal tissue sample, 200 ml of jugular venous blood were collected into a sterile bottle containing either sodium heparin or acid citrate dextrose solution and used for isolation of T lymphocytes. Following blood collection, 25 mg of PGF2{alpha} (Lutalyse) were administered to cows (n = 4) to induce luteal regression. Biopsy procedures and blood collection were also performed on control cows (n = 4) that received no PGF2{alpha} following these initial surgical procedures. These animals served as controls to determine the effect of the biopsy procedure on function of the CL and luteal cell-stimulated T lymphocyte proliferation. A second blood sample was collected from all cows 8 h following the biopsy procedure to determine concentrations of progesterone in plasma. The remaining luteal tissue was then removed transvaginally, and a second 200 ml of jugular venous blood were collected for isolation of T lymphocytes. Handling of animals and surgical procedures were conducted according to protocols approved by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University (Animal Use Protocol 99-AG002).

Dissociation of luteal tissue was performed according to procedures described previously [13]. Briefly, luteal tissue was minced and placed in 24 mM Hepes-buffered Ham F-12 culture medium containing 0.5% BSA, 20 µg/ml of gentamicin, and collagenase (2000 U/g tissue). After 1 h of dissociation, dispersed cells were decanted, and a second dissociation was performed on the remaining tissue. Mild centrifugation was used to pellet dispersed cells. Dissociation media were then removed, and cells were resuspended in serum-free RPMI 1640. This wash procedure was repeated a total of three times to remove BSA and collagenase and also to decrease the number of nonsteroidogenic cells. Following the final wash, cells were resuspended in RPMI 1640 containing 10% fetal calf serum. A small aliquot of the dispersed cell suspension was diluted in Trypan blue to determine cell viability, and cells were counted in a hemocytometer.

Isolation of T Lymphocytes

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected via jugular venipuncture at the time of biopsy and CL removal. The lymphocyte-rich white blood cell layer was collected following centrifugation of whole blood, and PBMCs were isolated by centrifugation through Ficoll-Paque Plus.

For cocultures of luteal cells and T lymphocytes collected before and after in vivo PGF2{alpha} administration, T lymphocytes were isolated according to procedures described previously [11]. Briefly, 1 mg of rabbit anti-bovine IgG was loaded onto Cellect bovine T lymphocyte immunocolumns. Columns were then rinsed with ice-cold PBS, and 75 x 106 PBMCs were added to columns in 1 ml of cold PBS. In this procedure, B lymphocytes are retained in the column, whereas T lymphocytes pass through. T lymphocytes that passed through the columns were collected in 5–7 ml of eluate. Viability and numbers of T lymphocytes were determined by Trypan blue staining and counting in a hemocytometer.

For coculture experiments to evaluate the effects of progesterone on T lymphocyte proliferation, an isolation procedure to improve the yield of T lymphocytes was devised using the MACS Cell Separation System (Miltenyi Biotec, Inc., Auburn, CA). Acid citrate dextrose (ACD)-A was used to prevent coagulation of whole blood (1:4, ACD-A:whole blood). White blood cell layers were collected following centrifugation, and PBMCs were isolated by centrifugation through Ficoll-Paque Plus. T lymphocytes were then separated from PBMCs by depletion of class II MHC-positive cells using the MACS Cell Separation System. Briefly, PBMCs (75 x 106) were incubated with 1 µg/ml each of mouse anti-bovine class II MHC antibodies TH14B, TH81A5, and H42A (VMRD, Pullman, WA), washed, and then labeled with rat anti-mouse IgG2a+b microbeads (Miltenyi Biotec). Cells were subsequently passed through a MACS MS+ column, and unlabelled cells were collected. This separation procedure yielded a population of cells that was approximately 96% positive for the T lymphocyte receptor-associated cell surface complex CD3 (as determined by immunofluorescent labeling with anti-CD3 antibody MM1A [VMRD]).

Coculture of Luteal Cells and T Lymphocytes

Experiment 1: Effect of PGF2{alpha} on luteal cell-stimulated T lymphocyte proliferation Coculture of luteal cells and T lymphocytes was performed as described previously [11]. Luteal cells were treated with 50 µg/ml of mitomycin C to prevent proliferation of luteal cells in culture. Cocultures of luteal cells and T lymphocytes from the same animal were arranged as follows: Luteal cells and T lymphocytes collected before administration of PGF2{alpha} were cultured together [LC(b)+TC(b)]; luteal cells collected before PGF2{alpha} administration were cultured with T lymphocytes collected after PGF2{alpha} administration [LC(b)+TC(a)]; luteal cells collected after administration of PGF2{alpha} were cultured with T lymphocytes collected before PGF2{alpha} administration [LC(a)+TC(b)]; and luteal cells and T lymphocytes collected after PGF2{alpha} were cultured together [LC(a)+TC(a)]. Additionally, luteal cells and T lymphocytes collected before and after in vivo administration of PGF2{alpha} were cultured separately to determine the amount of proliferation attributable to each cell type individually.

Experiment 2: Effect of progesterone on luteal cell-stimulated T lymphocyte proliferation To determine the effect of progesterone on luteal cell-stimulated T lymphocyte proliferation, CL were removed from cows (n = 4) on Days 10–12 of the estrous cycle and dissociated as described previously [13]. Cocultures of luteal cells and T lymphocytes were carried out in the absence or presence of 50 µg/ml of aminoglutethimide, an inhibitor of the cytochrome P450 side-chain cleavage enzyme. This concentration of aminoglutethimide has previously been shown to inhibit progesterone synthesis by cultured bovine luteal cells [14]. Additionally, increasing concentrations of exogenous progesterone were added to cocultures in the presence of aminoglutethimide. Progesterone (4-pregnen-3,20-dione; Steraloids, Wilton, NH) was solubilized in 100% ethanol and then added to cocultures in the presence of aminoglutethimide. Final concentrations of progesterone in cultures were 0.05, 0.5, and 5.0 µM and represent both physiological (0.05 and 0.5 µM) and pharmacological (5.0 µM) concentrations; these concentrations have been used previously to determine the effects of progesterone on cultured bovine luteal cells [15]. Equal volumes of 100% ethanol were added to untreated cultures to control for effects of the vehicle on T lymphocyte proliferation.

Coculture and T lymphocyte proliferation assay procedures For all coculture experiments, 3.2 x 104 luteal cells were placed in culture with 1.0 x 105 T lymphocytes in RPMI 1640 containing 10% heat-inactivated fetal calf serum, 25 mM HEPES, 2 mM L-glutamine, 100 IU of penicillin, and 100 µg/ml of streptomycin in the presence of 1 µg/ml of staphylococcal enterotoxin B. These coculture conditions were optimized for measurement of luteal cell-stimulated T lymphocyte proliferation in a previous study [11]. Cocultures were performed in a humidified atmosphere of 5% CO2 in air at 37°C for 72 h. During the last 6 h of culture, 0.5 µCi [3H]thymidine was added to each well to measure cellular proliferation. At the end of the 72-h culture period, culture plates were frozen at -80°C. Cells were subsequently harvested using a semiautomatic cell harvester (Skatron Instruments, Sterling, VA), and incorporation of [3H]thymidine into the cellular DNA (a measure of cell proliferation) was determined by liquid scintillation counting as described previously [11].

Progesterone-Receptor mRNA

Peripheral blood mononuclear cells were isolated from cows before and 8 h following administration of a luteolytic dose of PGF2{alpha} during the midluteal phase (Days 9–12 of the estrous cycle) as described above. After centrifugation through Ficoll-Paque Plus, the PBMC fraction was isolated and washed three times with ice-cold PBS, and total RNA was extracted using TRIzol reagent according to the manufacturer's specifications. Total RNA was treated with RNase-free DNase I (Roche Molecular Biochemicals, Mannheim, Germany) to eliminate DNA contamination, re-extracted with a 24:25:1 mixture of phenol:chloroform:isoamyl alcohol, and precipitated with 100% ethanol. Following precipitation, RNA was reconstituted in RNase-free water, and concentrations of total RNA were determined spectrophotometrically.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect progesterone-receptor mRNA in total RNA from PBMCs. Oligonucleotide primers specific for bovine progesterone-receptor mRNA (forward primer, 5'-CCGTAAGCCAGAGAATCACTT-3'; reverse primer, 5'-TTATGATGACTCCTTCATCCGC-3'; Qiagen Operon, Alameda, CA) have been described and used previously to amplify a 380-base pair (bp) progesterone-receptor cDNA fragment from bovine luteal tissue [16]. Bovine luteal tissue was therefore used as a source of positive control RNA in the present study. An 854-bp glyceraldehyde-3-phosphate dehydrogenase cDNA fragment was amplified in all samples using ovine specific primers described previously [17] to confirm the integrity of all RNA samples. Total cellular RNA (1 µg) was reverse transcribed using random hexamer primers and Superscript II reverse transcriptase according to the manufacturer's protocol. Following RT, PCR was performed for a total of 45 cycles under the following conditions: denaturing, 94°C for 30 sec; annealing, 55°C for 30 sec; and extension, 72°C for 60 sec. Following PCR, amplification products were electrophoretically separated on 1.5% agarose gels and visualized with ethidium bromide.

Statistical Analysis

Differences in mean plasma progesterone concentrations before and after in vivo administration of PGF2{alpha} were determined using a paired t-test. Because of a high amount of variability in T lymphocyte proliferation between animals in the luteal biopsy experiment, a proliferation index was used to determine the degree of T lymphocyte proliferation in cocultures of luteal cells and T lymphocytes collected before and after PGF2{alpha} administration. This proliferation index was determined by dividing the cell counts in luteal cell-T lymphocyte cocultures by the sum of counts from cultures of the individual cell types alone. Differences in the mean proliferation indices were determined by one-way analysis of variance, and Fisher least significant difference test was used to determine differences between specific means. In cocultures conducted to determine the effects of progesterone on luteal cell-stimulated T lymphocyte proliferation, T lymphocyte proliferation was very uniform between animals, and raw cell counts (rather than a proliferation index) were used for data analysis. Differences in mean cell counts from coculture experiments were determined using one-way ANOVA and Fisher least significant difference test. Differences between specific means were considered to be significantly different at P < 0.05. All statistical procedures were performed using the SigmaStat 2.0 software package (Jandel Scientific, San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Administration of a luteolytic dose of PGF2{alpha} reduced plasma progesterone concentrations by approximately 50% (9.8 ± 0.5 ng/ml vs. 4.6 ± 0.7 ng/ml, P < 0.05) within 8 h. The results of coculture experiments using luteal cells and T lymphocytes collected before or 8 h after PGF2{alpha} administration are depicted in Figure 1. When compared to luteal cells collected before administration of PGF2{alpha}, luteal cells collected 8 h after in vivo administration of PGF2{alpha} stimulated a greater (P < 0.05) degree of T lymphocyte proliferation when cultured with T lymphocytes collected before administration of PGF2{alpha} [LC(b)+TC(b) vs. LC(a)+TC(b)] (Fig. 1). Enigmatically, a similar effect was not observed when cocultures were performed using T lymphocytes collected after administration of PGF2{alpha}. From these results, it can be inferred that administration of a luteolytic dose of PGF2{alpha} enhances the ability of luteal cells to stimulate T lymphocyte proliferation in vitro.



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  		FIG. 1. Proliferation of T lymphocytes in luteal cell-T lymphocyte cocultures. Cultures were carried out with luteal cells and T lymphocytes collected before administration of a luteolytic dose of PGF2{alpha} [LC(b)+TC(b)], luteal cells collected after and T lymphocytes collected before administration of a luteolytic dose of PGF2{alpha} [LC(a)+TC(b)], luteal cells collected before and T lymphocytes collected after administration of a luteolytic dose of PGF2{alpha} [LC(b)+TC(a)], and luteal cells and T lymphocytes collected after administration of a luteolytic dose of PGF2{alpha} [LC(a) + TC(a)]. Data represent replicate experiments using four different animals. Different letters denote significant differences (P < 0.05)

To examine the effect of the luteal biopsy procedure on luteal function and luteal cell-stimulated T lymphocyte proliferation, biopsy procedures identical to those carried out on experimental animals were performed on four control animals to which PGF2{alpha} was not administered after initial collection of luteal tissue. Because no PGF2{alpha} was administered to cows for this experiment, T lymphocytes were collected only before the biopsy procedure. The results of this experiment are depicted as a change in proliferation index, which was used to determine whether a change in T cell proliferation occurred following the biopsy procedure. The change in proliferation index was calculated by dividing the proliferation index of cocultures containing luteal cells and T cells collected before biopsy by the proliferation index of cocultures containing luteal cells collected after biopsy and T cells collected before biopsy. Using this calculation, an increase in the proliferation index following luteal biopsy, which indicates an increase in the ability of luteal cells to stimulate T lymphocyte proliferation, would result in a number greater than one, whereas a lack of change in the proliferation index after biopsy, which indicates a lack of change in the ability of luteal cells to stimulate T lymphocyte proliferation, would result in a number less than or equal to one. Using this calculation has allowed us to compare the ability of luteal cells to stimulate T cells collected before or after the biopsy procedure. The results of this experiment are shown in Table 1. In two of the four control animals (R641 and R840), plasma progesterone concentrations declined by 53% and 34%, respectively, during the 8-h period following the biopsy procedure. Luteal cell-stimulated T lymphocyte proliferation increased following the biopsy procedure in both of these animals. Conversely, in the two other control animals (R644 and R839), no change was found in plasma progesterone concentrations following the biopsy procedure, nor was any increase observed in the ability of luteal cells to stimulate T lymphocyte proliferation (Table 1). Combined with the results from the previous experiment, these results indicate that a decline in the ability of luteal cells to synthesize progesterone may enhance their ability to stimulate T lymphocyte proliferation.


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  		TABLE 1. Plasma progesterone concentrations and changes in T cell proliferation indices in control animals.a

To determine the effect of progesterone on luteal cell-stimulated T lymphocyte proliferation, luteal cells and T lymphocytes were cocultured in the presence of aminoglutethimide, an inhibitor of the cytochrome P450 side-chain cleavage enzyme, which blocks progesterone synthesis. In the presence of aminoglutethimide, luteal cell-stimulated T lymphocyte proliferation was greater as compared with control cultures (P < 0.05) (Fig. 2). To determine if this effect resulted specifically from the inhibition of progesterone synthesis by luteal cells, exogenous progesterone was added to luteal cell-T lymphocyte cocultures in the presence of aminoglutethimide. Exogenous progesterone in the presence of aminoglutethimide inhibited luteal cell-stimulated T lymphocyte proliferation in a dose-dependent manner (P < 0.05) (Fig. 2).



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  		FIG. 2. Effects of progesterone (P4) on luteal cell-stimulated T lymphocyte proliferation. Bars represent T lymphocyte proliferation (as measured by [3H]thymidine incorporation) in cocultures of luteal cells and T lymphocytes in the absence or presence of aminoglutethimide (AG) and in the presence of aminoglutethimide and varying concentrations of exogenous progesterone. Data represent replicate experiments using four different animals. Different letters denote significant differences (P < 0.05)

Figure 3 displays the results of an experiment in which RT-PCR was used to detect progesterone-receptor mRNA in RNA from PBMCs, which are rich in T lymphocytes, and from luteal tissue. A 380-bp cDNA fragment corresponding to a portion of progesterone-receptor mRNA was amplified from luteal tissue RNA as described previously [16]. Progesterone-receptor mRNA was not detected in RNA isolated from PBMCs collected before or following administration of PGF2{alpha} (Fig. 3). Therefore, it can be concluded that the inhibitory effect of progesterone on luteal cell-stimulated T lymphocyte proliferation was not mediated by progesterone receptors in the T lymphocytes.



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  		FIG. 3. RT-PCR amplification of progesterone receptor mRNA (top) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA (bottom) in total RNA from isolated PBMCs collected before (lanes 2–5) or 8 h after (lanes 6–9) administration of a luteolytic dose of PGF2{alpha} to cows. Lane 1: 100-bp DNA size markers; lane 10: luteal tissue RNA (positive control)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, when cocultured with T lymphocytes collected before in vivo administration of PGF2{alpha}, luteal cells collected following administration of PGF2{alpha} exhibited a greater capacity to stimulate T lymphocyte proliferation compared with luteal cells collected before PGF2{alpha} administration. Oddly, in the present study, no difference in T lymphocyte proliferation was observed between cocultures of luteal cells and T lymphocytes collected before PGF2{alpha} and luteal cells and T lymphocytes collected after PGF2{alpha} [Fig. 1, LC(b)+TC(b) vs. LC(a)+TC(a)]. These results differ from those of a previous study [11] in which T lymphocyte proliferation was greater in luteal cell-T lymphocyte cocultures when both cell types were collected 8 h after PGF2{alpha} administration. Use of the biopsy procedure in the present study to obtain luteal tissue before administration of PGF2{alpha} is one obvious difference between the two studies. This procedure may account for the observed difference between the results of the present and of the previous study [11], but the mechanism responsible is not clear given the available data. This discrepancy warrants further investigation. However, the results of the previous study [11] clearly demonstrate that T lymphocyte proliferation is greater in cocultures of luteal cells and T lymphocytes collected following in vivo administration of PGF2{alpha}. In the present study, proliferation of T lymphocytes collected before administration of PGF2{alpha} was more potently stimulated by luteal cells collected following PGF2{alpha} administration as compared with luteal cells collected before administration of PGF2{alpha}. It can therefore be concluded that administration of PGF2{alpha} in vivo results in changes in luteal cells that enhance their ability to stimulate T lymphocyte proliferation in vitro.

To determine whether the biopsy procedure used in the present study could affect luteal function and the ability of luteal cells to stimulate T lymphocyte proliferation, an experiment was performed in which surgical procedures were carried out on animals to which PGF2{alpha} was not administered. In two of these four animals, the luteal biopsy procedure did not affect plasma progesterone concentrations, nor was the ability of luteal cells to stimulate T lymphocyte proliferation affected. However, in the other two animals, the luteal biopsy procedure caused a decline in plasma progesterone concentrations, and the ability of luteal cells to stimulate T lymphocyte proliferation increased. Therefore, it was speculated that reduction in progesterone production by luteal cells, whether induced in the present study by a luteolytic dose of PGF2{alpha} or in response to the luteal biopsy procedure, could account for the enhanced ability of luteal cells to stimulate T lymphocyte proliferation.

To determine whether a decrease in progesterone synthesis by luteal cells could account, at least in part, for the increases in T lymphocyte proliferation observed following in vivo PGF2{alpha} administration in the present study, the effect of progesterone on luteal cell-stimulated T lymphocyte proliferation was examined. Ideally, in the present study, concentrations of progesterone in luteal cell-T lymphocyte coculture medium would have been measured at the end of culture in conjunction with measurement of T lymphocyte proliferation (as determined by [3H]thymidine incorporation). This would have allowed us to determine directly whether a correlation existed between the concentration of progesterone in coculture medium and proliferation of T lymphocytes in luteal cell-T lymphocyte cocultures. However, the cell-harvesting procedure employed to determine [3H]thymidine incorporation in the present study was incompatible with removal of culture medium for determination of progesterone concentration. Instead, an experiment was performed in which exogenous progesterone was added to determine the effect of increasing concentrations of progesterone on luteal cell-stimulated T lymphocyte proliferation. In the presence of aminoglutethimide (an inhibitor of the cytochrome P450 side-chain cleavage enzyme) at a concentration previously shown to inhibit lipoprotein-stimulated progesterone production by bovine luteal cells [14], luteal cell-stimulated T lymphocyte proliferation was greater compared with cocultures in the absence of aminoglutethimide. This suggests that progesterone synthesis by luteal cells affects T lymphocyte proliferation in this coculture system. Furthermore, when progesterone, in a range representing both physiological (0.05 and 0.5 µM) and pharmacological (5.0 µM) concentrations [15], was added to cultures in the presence of aminoglutethimide, a dose-dependent inhibition of T lymphocyte proliferation was observed with increasing concentrations of exogenous progesterone. These data indicate that the progesterone produced by luteal cells can inhibit luteal cell-stimulated T lymphocyte proliferation in vitro. Because in vivo administration of PGF2{alpha} inhibits lipoprotein- and LH-stimulated progesterone production by bovine luteal cells in vitro [18], it is likely that, in the present study, luteal cells collected after administration of PGF2{alpha} synthesize less progesterone and, therefore, that less progesterone accumulates in the culture medium. It thus is plausible that lower concentrations of progesterone accumulated in cocultures containing luteal cells collected after PGF2{alpha} administration, allowing a greater degree of T lymphocyte proliferation.

In the present study, we cannot discount the possibility that, in addition to progesterone, other luteal cell products may affect luteal cell-stimulated T lymphocyte proliferation. In addition to progesterone, bovine luteal cells produce PGE2, PGF2{alpha}, and prostacyclin [1, 19, 20], and luteal endothelial cells likely produce these PGs as well. The effect of prostacyclin on bovine lymphocytes is not known. However, PGF2{alpha} is without effect on the proliferation of bovine lymphocytes [21, 22], and PGE2 has been shown to inhibit proliferation of bovine lymphocytes in vitro [22]. Therefore, it is likely that PGE2 synthesized by the luteal cells in this culture system can also inhibit luteal cell-stimulated T lymphocyte proliferation. However, previous studies [12, 23–26] have demonstrated an inhibitory effect of progesterone on lymphocyte proliferation in several species, including the cow, and in the present study, exogenous progesterone does-dependently inhibited luteal cell-stimulated T lymphocyte proliferation. The inhibitory effect of progesterone on T lymphocyte proliferation in the present study does not appear to be mediated via progesterone receptors in T lymphocytes, because progesterone-receptor mRNA was not detected by RT-PCR in total RNA isolated from PBMCs, which are rich in T lymphocytes. In previous studies, the progesterone-receptor antagonists RU38486 and RU43044 were unable to reverse the inhibitory effects of progesterone on lymphocyte proliferation [26, 27]. These results support an effect of progesterone on lymphocyte proliferation despite the apparent lack of progesterone receptors in bovine lymphocytes. Collectively, it can be concluded that progesterone produced by luteal cells in this coculture system can inhibit luteal cell-stimulated T lymphocyte proliferation via progesterone receptor-independent actions on T lymphocytes.

Because T lymphocyte proliferation observed in the present study is dependent on the presence of class II MHC molecules on luteal cells, it is tempting to speculate that progesterone may cause a decrease in expression of class II MHC molecules by luteal cells. Progesterone receptors are present in small and large luteal cells and luteal endothelial cells, and each of these cell types is responsive to progesterone in vitro [17, 28]. However, in a previous study, progesterone was without effect on IFN-{gamma}-induced expression of class II MHC molecules by cultured bovine luteal cells [29]. It is therefore unlikely that the inhibitory effect of progesterone on luteal cell-stimulated T lymphocyte proliferation results from a progesterone-induced decline in expression of cell surface class II MHC molecules by luteal cells. The possibility of a more subtle effect of progesterone on the ability of luteal cells to stimulate T lymphocyte proliferation cannot be ruled out, however. Class II MHC molecules present antigenic peptides to T lymphocytes, and changes in the array of antigenic peptides presented to T lymphocytes in the context of class II MHC molecules can alter the ability of a cell expressing class II MHC molecules to induce T lymphocyte activation [30, 31]. In the case of class II MHC, changes in expression of intracellular proteins required to process the peptides presented by class II MHC molecules can alter the array of peptides bound to class II MHC molecules [32–34]. It is possible that progesterone affects expression of genes encoding intracellular proteins responsible for processing of peptides presented by class II MHC molecules, independent of overall changes in expression of class II MHC. Such changes could result in alterations in the array of peptides bound to MHC molecules on luteal cells. This could in turn enable luteal cells to stimulate T lymphocyte activation following exposure to PGF2{alpha}.

In conclusion, administration of PGF2{alpha} in vivo induces changes in luteal cells that enhance their ability to stimulate T lymphocyte proliferation in an in vitro coculture system. In the present study, exogenous progesterone inhibited luteal cell-stimulated T lymphocyte proliferation, indicating that a decrease in the ability of luteal cells to secrete progesterone following exposure to PGF2{alpha} may, at least in part, account for this effect. The inhibitory effect of progesterone occurs despite the apparent absence of progesterone receptors in bovine T lymphocytes, because progesterone-receptor mRNA was undetectable in peripheral blood lymphocytes. The effects of progesterone may be manifested directly on the lymphocytes, in a receptor-independent fashion, and/or through modulation of peptide presentation in the context of class II MHC molecules by the luteal cells.


   FOOTNOTES
 
1 Supported in part by NIH grant HD37550; salaries and research support also provided by state and federal funds appropriated. Back

2 Correspondence: Joy L. Pate, Department of Animal Sciences, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Ave., Wooster, OH 44691. FAX: 330 263 3949; pate.1{at}osu.edu Back

3 Current address: Anatomy and Cell Biology Unit, The University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160 Back

Received: 25 March 2003.

First decision: 14 April 2003.

Accepted: 21 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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