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Biology of Reproduction 61, 1017-1022 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Tumor Necrosis Factor {alpha} Receptors in Microvascular Endothelial Cells from Bovine Corpus Luteum1

Kiyoshi Okuda2,a, Ryosuke Sakumotoa, Yoshihisa Uenoyamaa, Bajram Berishab, Akio Miyamotob, and Dieter Schamsb

a Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan b Institute of Physiology, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is sufficient evidence to prove that tumor necrosis factor {alpha} (TNF{alpha}) modulates bovine corpus luteum (CL) function. Our previous study demonstrated that functional TNF{alpha} receptors are present on luteal cells in bovine CL throughout the estrous cycle. The purpose of the present study was to identify the presence of functional TNF{alpha} receptors on the microvascular endothelial cells derived from developing bovine CL. TNF{alpha} receptors were analyzed by a radioreceptor assay using 125I-labeled TNF{alpha} on two types of cultured endothelial cells. One has a cobblestone appearance (CS cells), and the other has a tube-like structure (TS cells). 125I-Labeled TNF{alpha} binding was maximal after incubation for 30 h at 37°C, and the specificity of binding was confirmed. A Scatchard analysis showed the presence of two binding sites (high- and low-affinity) for TNF{alpha} receptors on both CS and TS cells. The dissociation constant (Kd) values and concentrations of the high-affinity binding sites for TNF receptors were similar for CS and TS cells. However, Kd values and concentrations of the low-affinity binding sites in CS cells were significantly higher than those in TS cells (P < 0.05 or lower). The expression of TNF receptor type 1 (TNF-RI) mRNA was determined in both cell types. Furthermore, TNF{alpha} significantly stimulated prostaglandin E2 and endothelin-1 secretion by both CS and TS cells (P < 0.05 or lower). These results indicate the presence of two types of TNF receptors and the expression of TNF-RI mRNA in the endothelial cells derived from bovine CL, and suggest that TNF{alpha} plays two or more roles in regulating the secretory function of the endothelial cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor {alpha} (TNF{alpha}) is usually described as a tumoricidal cytokine, which is produced by activated macrophages [1]. It has been assumed that TNF{alpha} plays one or more roles as one of the local regulators (a variety of hormones, cytokines, growth factors, etc.) in regulating the female reproductive systems including ovary, uterus, and corpus luteum (CL) function in a variety of species [2–4]. It has been demonstrated that TNF{alpha} affects gonadotropin-induced steroidogenesis and protein secretion by bovine granulosa [5], theca [6], and luteal cells [7] in vitro. Furthermore, our recent study provided evidence that functional TNF receptors are present in bovine CL throughout the estrous cycle, and suggested that the actions of TNF{alpha} in regulating bovine CL function are mediated by its specific receptors [8]. However, since we used membrane homogenates that were obtained from whole CL for the radioreceptor assay in the previous study, it was not possible to say which cell types (large or small luteal cells, endothelial cells, fibroblasts, etc.) have TNF receptors. Recently, it has been shown that specific binding sites for TNF{alpha} were present on both granulosa and theca cells in bovine ovary [9] and that TNF{alpha} clearly stimulated prostaglandin (PG) F2{alpha} and PGE2 secretion by highly purified bovine luteal cells [8, 10]. On the basis of these latest findings, we assume that TNF receptors are present at least on luteal cells in bovine CL, although it is still unclear whether CL cells other than luteal cells contain TNF receptors.

On the other hand, it is well recognized that endothelial cells constitute the major proportion (53.5%) of the bovine CL [11]. The microvascular endothelial cells in several tissues produce PGE2 [12] and endothelin-1 (ET-1) [13]. These hormones have been demonstrated to affect steroidogenesis of bovine CL in vitro [14–16]. The presence of high-affinity binding sites for TNF{alpha} has been shown in bovine aortic endothelial cells [17]. In addition, it has been demonstrated that TNF{alpha} stimulated phospholipase A2 activity in bovine aortic endothelial cells [18], which suggests that functional TNF receptors are also present on endothelial cells derived from CL. Hence, in addition to TNF{alpha}'s action on the function of the luteal cells, we hypothesized that it also acts as a paracrine regulator in regulating the function of the endothelial cells in bovine CL via its specific receptors.

Therefore, in the present study two types of cultured microvascular endothelial cells that were obtained from developing bovine CL [19] (one expressing a cobblestone appearance and the other possessing a tube-like structure) were examined for the expression of TNF receptor type 1 (TNF-RI) mRNA by reverse transcription (RT) polymerase chain reaction (PCR) analysis, and the TNF{alpha} binding characteristics of these cells were investigated by radioreceptor assay. Furthermore, the possible effects of TNF{alpha} on PGE2 and ET-1 secretion by cultured endothelial cells were studied.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture of Microvascular Endothelial Cells

Cytokeratin-negative endothelial cells, type 3, derived from the microvascular bed of the developing bovine CL [19–21] were kindly provided by Dr. Spanel-Borowski (University of Leipzig, Germany). These cytokeratin-negative cells are known to occur in two forms in confluent culture. One form has a cobblestone appearance (CS cells), and the other form spontaneously expresses a tube-like structure (TS cells). The CS and TS cells were separately cultured for 8 passages. Both CS and TS cells were then stored in liquid nitrogen. These cells retain a stable form during culture for at least 17 passages (data not shown). Only the cells at 11 passages were used in the present study.

The cells were cultured in flasks (80 cm2, 260 ml, #178905; Nunc, Roskilde, Denmark) until they were confluent. The flasks were pre-coated with 1% collagen type I (Vitrogen 100; Collagen Corp., Palo Alto, CA) for 2 h at 37°C. For the culture medium, Dulbecco's modified Eagle's medium (DMEM; #31600-026; Gibco BRL Life Technologies, Grand Island, NY) and Ham's nutrient mixture F-12 (Ham's F-12; #21700-026; Gibco) were mixed 1:1 (v:v) with 15 mM HEPES (#H0763; Sigma Chemical Co., St. Louis, MO), 22 mM NaHCO3 (#S5761; Sigma), 5% fetal calf serum (FCS; #S0115; Biochrom Beteiligungs GmbH & Co., Berlin, Germany), and 20 mg/L gentamicin (#G1264; Sigma). When the cells were confluent, 0.02% trypsin (1:250; #T0646; Sigma) solution was added to the cells for 7 min at 37°C. The cells were removed and counted with a hemocytometer and assessed for viability by trypan blue dye exclusion. Cell viability was higher than 90%.

Experiment 1. Two types of microvascular endothelial cells were examined for the presence of TNF{alpha} receptors by a radioreceptor assay using a 125I-labeled recombinant human TNF{alpha} (Lot. No. HF-13; kindly donated by Dainippon Pharmaceutical Co. Ltd., Osaka, Japan). The cells were resuspended in fresh culture medium containing 5% FCS and were plated in 24-well cluster dishes (Falcon #3047; Becton Dickinson & Co., Lincoln Park, NJ; 300 000 cells/well) in a humidified atmosphere of 5% CO2 in air at 37°C. Confluence of the cells was generally observed after 4–5 days in culture.

Experiment 2. Cells prepared as described above were plated in 48-well cluster dishes (#3548; Costar, Cambridge, MA; 150 000 cells/well) in a humidified atmosphere of 5% CO2 in air at 37°C. Confluence of the cells was generally observed after 4–5 days in culture. When the cells were confluent, they were washed two times with 250 µl of fresh culture medium containing 0.1% BSA (#A7888; Sigma) and 20 mg/L gentamicin. After being washed, the cells were incubated in fresh medium containing 0.1% BSA, 10 µM arachidonic acid (#A8798; Sigma), 2 mg/L insulin (#977420; Boehringer Mannheim GmbH, Mannheim, Germany), 5 mg/L transferrin (#T3400; Sigma), 5 µg/L sodium selenite (#S5261; Sigma), 20 mg/L gentamicin, and varying concentrations of TNF{alpha} (0.06–3 nM) for 24 h at 37°C. After incubation, conditioned media were collected in tubes with 5 µl of a stabilizer (0.3 M EDTA, 1% aspirin [#A2093; Sigma], pH 7.3) and stored at -20°C until assayed for PGE2 and ET-1.

DNA Assay

At the end of each experiment, the DNA content of the endothelial cells was estimated spectrophotometrically as described by Labarca and Paigen [22]. Briefly, the cells were washed 2 times with 250 µl of phosphate-saline buffer (50 mM NaH2PO4, 140 mM NaCl, 2 mM EDTA; pH 7.4) and completely destroyed by ultrasonication for 20 sec. The samples and standard were dispensed in a 96-well plate (#7655077; Greiner GmbH, Frickenhausen, Germany), and then 40 µl of bis-benzimide (8.43 µM, #B2883; Sigma) was added into each well of the plate. After 10 min at 4°C, the fluorescence was evaluated by FLUOROSKAN II (Flow Laboratories GmBH, Meckenheim, Germany). The DNA from calf thymus (#D3664; Sigma) was used as a standard, and the standard curve was determined for concentrations in the range from 0.09 to 12.5 µg/ml. DNA content of the cultured cells was not changed by TNF{alpha} treatment. However, since TNF{alpha} slightly reduced the DNA content of cultured cells, it was used to standardize the results.

RNA Isolation

Total RNA from bovine CL was isolated by the single-step method of Chomczynski and Sacchi [23] using TRIzol reagent (Gibco BRL, Gaithersburg, MD). Total RNA from endothelial cells was isolated using the NucleoSpin RNA kit (Macherey-Nagel, Dueren, Germany). Finally, RNA was dissolved in water and spectroscopically quantified at 260 nm. Aliquots were electrophoresed on a 1% denaturing agarose gel to verify the quantity and quality of RNA by ethidium bromide staining.

RT-PCR

Four micrograms of total RNA was used to generate single-strand cDNA in a 60-µl reaction mixture as described previously [24]. Conditions for enzymatic amplification were optimized for each PCR as follows: the TNF-RI PCR contained 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.6 µM of each primer, and 0.5 units of thermostable polymerase PrimeZyme (Biometra, Göttingen, Germany) to 5 µl cDNA (final volume 25 µl). The ubiquitin PCR was performed under the same conditions as those for TNF-RI, but a higher concentration of primer (1.5 µM) was used. PCR conditions for TNF-RI consisted of a denaturation step at 94°C for 2 min followed by 28 cycles of 94°C for 1 min and 60°C for 1 min, followed by an additional elongation step at 72°C for 2 min. PCR conditions for the housekeeping gene ubiquitin consisted of a denaturation step at 94°C for 2 min followed by 22 cycles of 94°C for 45 sec, 55°C for 45 sec, and 72°C for 45 sec, followed by an additional elongation step at 72°C for 2 min. To determine the optimal quantity of reverse transcriptase needed for PCR and to verify that the cDNA product depended on the mRNA transcript used for a template, varying quantities of transcriptase were used in the PCR reaction. The RT product from 3 µl was in the linear range of these amounts and produced a visible band. To exclude the possibility of amplification of genomic DNA, all experiments included reactions in which the RT enzyme or cDNA template was omitted. As a negative control, water was used instead of RNA for the RT-PCR to exclude any contamination from buffers and tubes.

The primers encoding the bovine sequences were designed using the EMBL database or were used as described elsewhere and were commercially synthesized (Amersham-Pharmacia, Freiburg, Germany). The primers were selected using the "Husar" online software package in Heidelberg (http://genome.dkfz-heidelberg.de). The primers 5'-CACCACCACCATCTGCTT-3' (forward primer) and 5'-TCTGAACTGGGGTGCAGA-3' (reverse primer) were used to amplify the TNF-RI gene. The primer sequences of ubiquitin (forward, 5'-ATGCAGATCTTTGTGAAGAC-3'; reverse, 5'-CTTCTGGATGTTGTAGTC-3') were designed as described by Gabler et al. [25].

Aliquots of the PCR reaction products (5 µl) were added to 1 µl bromphenol blue glycerin and fractionated by electrophoresis through a 1.5% agarose gel containing ethidium bromide in a constant 60-V field. To determine the length of the products, a mass ladder and 100-base pair (bp) marker were used. The ethidium bromide-stained gels were evaluated by a video documentation system (Amersham-Pharmacia). The band intensities were analyzed by computerized densitometry using the image master program (Amersham-Pharmacia). To verify each PCR product, double-strand sequencing was performed directly or after subcloning (TopLab, Munich, Germany).

Radioreceptor Assay

TNF{alpha} was iodinated with carrier-free 125I-Na (Iodine-125; #IMS 30; Amersham International plc., Buckinghamshire, UK) by Iodo-gen iodinating reagent (1.2 µg; #28600; Pierce Chemical Co., Rockford, IL) as described previously [26]. The specific activity of 125I-labeled TNF{alpha} ranged between 530 and 540 Ci/mmol.

When the cells were confluent, they were washed 2 times with 500 µl of modified Ca2+- and Mg2+-free Hanks' Balanced Salt Solution (mHBSS; #H2387; Sigma). The cells were then incubated with 50 000 disintegrations per minute (0.1 nM) 125I-labeled TNF{alpha} and 0–42 nM unlabeled TNF{alpha}. All incubations were performed in 560 µl of mHBSS containing 10 mM MgCl2 and 0.1% BSA (#11930; Serva Feinbiochemica GmbH & Co., Heidelberg, Germany) for 30 h at 37°C. The incubation was terminated by rapid washing of the cells with ice-cold mHBSS. After being washed two times, the cells were removed from the cluster dishes with 250 µl of 0.02% trypsin, and the bound radioactivity was counted in a gamma-counter (Pharmacia-Wallac 1282; Compugamma CS, Turku, Finland) at an efficiency of 82%. The difference in the 125I-TNF{alpha} binding in the presence and the absence of unlabeled TNF{alpha} (42 nM) was used to calculate the specific binding.

To determine the ligand specificity of the receptor, a competitive binding assay was performed with TNF{alpha}, recombinant human interleukin-1{alpha} (IL-1{alpha}, Lot. No. HL-18; kindly donated by Dainippon Pharmaceutical Co.) and recombinant bovine interferon-{alpha} (IFN{alpha}; kindly donated by Novartis Pharmaceuticals Co., Basel, Switzerland).

PGE2 and ET-1 Determination

The concentrations of PGE2 in the culture medium were determined with an enzyme immunoassay (EIA) using peroxidase-labeled PGE2 (1:10 000 final dilution) and PGE2 antiserum (1:70 000 final dilution; kindly donated by Dr. Seiji Ito of the Kansai Medical University, Osaka, Japan). Cross-reactivities of the PGE2 antiserum, validated by comparing the inhibition of binding of peroxidase-labeled PGE2 to antiserum, were as follows: PGE2, 100%; PGE1, 18%; PGA1, 10%; PGA2, 4.6%; PGB2, 6.7%; PGD2, 0.13%; PGF2{alpha}, 2.8%; PGJ2, 14%; and 15-keto PGE2, 0.05%. The PGE2 standard curve ranged from 0.3 to 5000 nM, and the ED50 of the assay was 52.1 nM. The intra- and interassay coefficients of variation were 3.5% and 6.8%, respectively.

The concentrations of ET-1 were determined directly in the culture media by EIA as described previously [15]. ET-1 antiserum (1:10 000 final dilution) and biotin-labeled ET-1 (1:2000 final dilution) were used in the assay. The standard curve ranged from 3.9 to 2000 pM, and the ED50 of the assay was 180 pM. The intra- and interassay coefficients of variation were 8.7% and 12.6%, respectively.

Statistical Analysis

The data of the binding characteristics and the concentrations of ET-1 and PGE2 are shown as the mean ± SE of values obtained from separate experiments, each performed in triplicate. The statistical significance of differences between the control and the treated group was assessed by ANOVA followed by Fisher's protected least-significant differences (PLSD) procedure as a multiple-comparison test. The data obtained from the binding of TNF{alpha} to the endothelial cells were analyzed with the LIGAND program [27] using nonlinear iterative curve-fitting procedures [28]. The initial parameters were calculated by Scatchard analysis [29] and were then iteratively refined until the weighted sum of squares was minimized. The goodness of fit for the selected model was analyzed by a runs test. Different models (one or two binding sites) were compared using F-test statistics to determine whether a change in the model resulted in a significant reduction in the weighted sum of squares. The criteria for rejecting or accepting a particular model were based on the calculated probability values [27].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Receptor Assay Conditions and Binding Characteristics

The conditions for the radioreceptor assay on the endothelial cells were initially validated. It was confirmed that maximal binding was reached after 24 h at 37°C (Fig. 1a). On the basis of these results, further assays were carried out under these conditions.



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  		FIG. 1. a) Relationship between the binding of 125I-labeled TNF{alpha} and incubation time at 37°C (circles) or 20°C (squares) on cultured microvascular endothelial CS cells obtained from developing bovine CL. b) Competitive binding on cultured microvascular endothelial cells (black circles, CS cells; gray circles, TS cells) of 125I-labeled TNF{alpha} and unlabeled TNF{alpha} (circles), IL-1{alpha} (squares), and IFN{alpha} (triangles)

Figure 1b shows the competition curves of 125I-labeled TNF{alpha} with three cytokines in the cultured endothelial cells. The binding was highly specific for TNF{alpha}, whereas IL-1{alpha} and IFN{alpha} did not display any competition with 125I-labeled TNF{alpha}.

Scatchard plots of the competitive binding of 125I-labeled TNF{alpha} and unlabeled TNF{alpha} on both CS and TS cells were curvilinear (Fig. 2, a and b), indicating that there were two binding sites in each cell type. Mean values of the dissociation constant (Kd) and the concentration of these receptors were analyzed by the LIGAND program (Table 1). Kd values and concentrations of high-affinity binding sites were similar in both CS and TS cells. On the other hand, Kd values and concentrations of low-affinity binding sites of CS cells were significantly higher than those of TS cells (P < 0.05).



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  		FIG. 2. Scatchard plots for competitive binding of 125I-labeled TNF{alpha} and unlabeled TNF{alpha} on endothelial CS (a) and TS (b) cells, obtained from developing bovine CL. Different symbols represent the data of three separate experiments


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  		TABLE 1. 125I-TNF{alpha} binding affinities and concentrations of the high- and low-affinity binding sites of cultured endothelial cells obtained from bovine corpus luteum.*

Expression of mRNA for TNF-RI

Specific transcripts for TNF-RI were detected in cultured endothelial cells and bovine CL. The PCR product showed 100% homology to the known bovine genes after sequencing. To confirm the integrity of the mRNA templates and RT-PCR protocol, the housekeeping gene ubiquitin was examined in all samples. A representative sample for the ubiquitin-specific RT-PCR products (189 + 417 bp) is shown in Figure 3a. The relative signal intensities (in arbitrary units; n = 3) for PCR products specific for TNF-RI were assessed after correction based on the ubiquitin signal intensities. We repeated the PCR analyses using three sets of separate RNA samples and obtained similar results. A representative example for the TNF-RI RT-PCR is given in Figure 3b. There were clear differences in the expression between the samples (1.52 ± 0.14, 0.62 ± 0.05, and 0.42 ± 0.06 in CL, and CS and TS cells, respectively). The expression of TNF-RI was lower in endothelial cells than in CL.



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  		FIG. 3. Specific RT-PCR products for a) ubiquitin (189+417 bp) and b) TNF-RI (257 bp) separated by agarose gel electrophoresis. Lane 1) DNA mass ladder, 2) bovine luteal tissue, 3) CS cells, and 4) TS cells

Effects of TNF{alpha} on PGE2 Secretion by Cultured Endothelial Cells

TNF{alpha} stimulated PGE2 secretion by both CS and TS cells (P < 0.05, Fig. 4). The mean concentration of PGE2 in the culture media of CS cells (Fig. 4a) was 10 times higher than that of TS cells (9.20 ± 0.65 and 0.82 ± 0.04 pmol/µg DNA in CS and TS cells, respectively) (Fig. 4b).



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  		FIG. 4. Effects of TNF{alpha} on PGE2 (a and b) and ET-1 (c and d) secretion by two forms of endothelial cells obtained from developing bovine CL. The cells were exposed to TNF{alpha} in the final 24 h of culture. All values are expressed as a percentage of the control value. The concentrations of PGE2 in the control were 9.20 ± 0.65 or 0.82 ± 0.04 pmol/µg DNA in a) CS or b) TS cells, respectively. The concentrations of ET-1 in the control were 1.40 ± 0.13 or 0.66 ± 0.26 pmol/µg DNA in c) CS or d) TS cells, respectively. Different superscript letters indicate significant differences (P < 0.05 or lower), as determined by ANOVA followed by Fisher's PLSD as a multiple comparison test. Bars marked "ab" are not significantly different from either bars marked "a" or bars marked "b"

Effects of TNF{alpha} on ET-1 Secretion by Cultured Endothelial Cells

ET-1 secretion by both CS and TS cells also increased as a result of treatment with TNF{alpha} (P < 0.05, Fig. 4, c and d). The mean concentrations of ET-1 in the culture media of CS and TS cells without TNF{alpha} were 1.4 ± 0.13 and 0.66 ± 0.26 pmol/µg DNA, respectively, and these values were not significantly different.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study presents strong evidence for the existence of TNF{alpha} binding sites and TNF-RI mRNA expression in two different forms of endothelial cells derived from developing bovine CL. In addition, the fact that TNF{alpha} stimulated PGE2 as well as ET-1 secretion by the cultured endothelial cells used in this study confirmed that the receptors for TNF{alpha} on the cells are functional.

In the present study, the Scatchard analyses clearly demonstrated that two binding sites (high-affinity binding site, Kd = 0.2–1.4 nM; low-affinity binding site, Kd = 56.5–108 nM) for TNF{alpha} are present on both CS and TS cells. It is well known that there are two types of TNF receptors, i.e., TNF receptors 1 and 2 (p55 and p75, respectively), and that these receptors have different intracellular signaling pathways [30]. Furthermore, it has been shown that endothelial cells derived from human umbilical vein have both p55 and p75 receptors for TNF [31, 32]. Thus, the results of our binding test raise the possibility that these two receptors (p55 and p75) are expressed on endothelial cells in the bovine CL. Moreover, the concentrations of TNF{alpha} (0.6–3 nM) that increased PGE2 and ET-1 secretion in the present study are comparable with the affinity of high-affinity binding sites for TNF{alpha} on cultured endothelial cells that were found in the present study. Therefore, we assume that the stimulatory effects of TNF{alpha} on PGE2 and ET-1 secretion might be mediated at least by the high-affinity TNF{alpha} receptors present in endothelial cells.

With the technique of RT-PCR, we were able to demonstrate the expression of TNF-RI mRNA in the endothelial cells. The intensity of expression did not differ between CS and TS cells, in agreement with the data for binding affinities and concentrations of binding sites. When compared with the mRNA from total CL tissue, the intensity of expression was lower in endothelial cells. Although a direct comparison in the intensities of mRNA between freshly prepared CL and cultured endothelial cells is not possible, it might be speculated that the luteal cells are the main target cells for TNF{alpha} in the bovine CL.

Although the endothelial cells from bovine CL were well characterized morphologically [19–21], the function of the cells is still not understood. The present study demonstrated that the basal secretion of ET-1 from CS cells tended to be higher than that from TS cells. Moreover, the basal secretion of PGE2 from CS cells was 10 times higher than that of TS cells. The type 3 endothelial cells are thought to be derived from postcapillary venules [19]. Normally, cell growth (angiogenesis) occurs at the postcapillary venules. Moreover, tubular forms of the endothelial cells correspond to angiogenesis. In general, proliferating cells are thought to have poor ability to produce hormones. Thus, one might speculate that TS cells mainly participate in angiogenesis and have a poor ability to produce hormones, including PGE2.

The present study clearly demonstrated that PGE2 secretion by cultured endothelial cells was significantly stimulated by treatment with TNF{alpha}. It is well known that macrophages [1] and endothelial cells [33] are sources of TNF{alpha}, and that these cells infiltrate into newly formed CL concomitantly with vascular angiogenesis [34, 35]. Since it has been clearly demonstrated that both TNF{alpha} [36] and PGE2 [37] affect the proliferation of endothelial cells, we assume that TNF{alpha}, and PGE2 induced by TNF{alpha}, may play a role in inducing vascular angiogenesis as autocrine and/or paracrine regulators. In addition, PGE2 is known to stimulate progesterone production from bovine CL in vitro as a luteotropic agent [14, 38].

On the other hand, TNF{alpha} also stimulated ET-1 secretion by the cultured endothelial cells in this study. Since some previous studies showed that ET-1 has a luteolytic effect in bovine CL in vitro [15, 16], TNF{alpha}-induced ET-1 from the endothelial cells may act on luteal cells as a luteolytic agent in the CL concomitantly with endometrial PGF2{alpha}. Furthermore, it has been postulated that TNF{alpha} directly acts on luteal cell function at the time of luteolysis [2, 3]. For example, it has been clearly demonstrated that TNF{alpha} induces a significant increase in the expression of major histocompatibility (MHC) class 1 glycoproteins in cultured bovine luteal cells and that these glycoproteins are recognized by cytotoxic T cells in order for the T cells to devour the luteal cells [7]. Moreover, it was shown that TNF{alpha} induced apoptosis of cultured mouse luteal cells [39]. Thus, it could be assumed that TNF{alpha} affects the function of the endothelial cells as well as the luteal cells at the time of luteolysis to complete the luteal regression. However, the physiological roles of ET-1, which is secreted throughout the estrous cycle [40], have not been clearly defined. On the other hand, it has been well demonstrated that TNF{alpha} plays a role as a potent stimulator of luteal PGs including PGF2{alpha}, PGE2, and PGI2 [7, 8, 10]. The stimulatory effects of TNF{alpha} on luteal PGF2{alpha} and PGE2 production in the early luteal stage (Days 2–4) were much higher than those in later stages [10]. Moreover, these luteal PGs are known to stimulate progesterone production from bovine CL in vitro as luteotropic agents [14, 38, 41]. Since TNF{alpha} receptors in whole CL are present throughout the estrous cycle [8], it is possible that TNF{alpha} acts on bovine CL function as either a luteotropic or luteolytic agent depending on the stages of the estrous cycle.

In conclusion, the overall results of the present study indicate the presence of functional TNF{alpha} receptors (high- and low-affinity receptors) on the endothelial cells in bovine CL. Since our previous study demonstrated that functional TNF{alpha} receptors are present on bovine CL throughout the estrous cycle [8], these results strongly suggest that TNF{alpha} plays two or more roles in regulating bovine CL function as an autocrine and/or paracrine regulator throughout the estrous cycle.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Katharina Spanel-Borowski of the University of Leipzig for the endothelial cells; Dr. Seiji Ito of the Kansai Medical University for PGE2-antiserum; the Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan) for recombinant human TNF{alpha} and IL-1{alpha}; and the Novartis Pharmaceuticals Co. (Basel, Switzerland) for recombinant bovine IFN{alpha}.


   FOOTNOTES
 
1 This research was supported by a Grant-in-Aid for Scientific Research (No. 11460129) from the Ministry of Education, Science, Sports and Culture of Japan; the Japanese-German Cooperative Science Promotion Program of the Japanese Society for the Promotion of Science (JSPS); and the German Research Foundation (Scha 257/14-1). R.S. (No. 8763) and Y.U. (No. 6032) are Research Fellows of JSPS. A.M. is supported by Alexander von Humbolt Stiftung. Back

2 Correspondence. FAX: 81 86 251 8388; kokuda{at}cc.okayama-u.ac.jp Back

3 Current address: Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan. Back

Accepted: May 18, 1999.

Received: March 9, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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