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Biology of Reproduction 62, 1427-1435 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Article

Expression and Regulation of the CC-Chemokine Monocyte Chemoattractant Protein-1 in Rat Testicular Cells in Primary Culture1

Florence Aubrya, Cécile Habasquea, Anne-Pascale Satiea, Bernard Jégoua, and Michel Samson2,a

a GERM-INSERM U. 435, Université de Rennes I, Campus de Beaulieu, 35042 Rennes cedex, Bretagne, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testicular inflammation is classically observed in pathogenesis caused by infectious agents, environmental toxins, trauma, or autoimmune reactions and can lead to transitory or even permanent sterility. In these situations, a leukocyte infiltration is generally encountered. Macrophage inflammatory proteins (MIP)-1{alpha} and -1ß and monocyte chemoattractant protein-1 (MCP-1) are CC-chemokines involved in macrophage and lymphocyte chemoattraction. In the present study, using reverse transcription-polymerase chain reaction, Northern blot, and a specific ELISA, we investigated whether or not these chemokines are present within the testis and whether they are induced by a number of proinflammatory cytokines and lipopolysaccharides (LPS). MIP-1{alpha} and MIP-1ß were not detected in Sertoli cells, germ cells, peritubular cells, or Leydig cells. In contrast, MCP-1 mRNA and protein were found to be expressed by control isolated peritubular cells, and expression was markedly stimulated by interleukin-1{alpha} and-1ß (IL-1{alpha} and IL-1ß), tumor necrosis factor {alpha} (TNF-{alpha}), interferon {gamma}, and LPS. Leydig cells expressed MCP-1 when stimulated by IL-1ß. In contrast, MCP-1 was not found to be produced by Sertoli cells or germ cells as established by Northern blot and ELISA techniques. The kinetics of MCP-1 production by peritubular cells, as demonstrated by expression as early as 8 h poststimulation, are compatible with there being a rapid mobilization of these cells and this chemokine in an inflammatory process. Moreover, MCP-1 production by peritubular cells after half-maximal stimulation by LPS, TNF-{alpha}, and IL-1ß (2 pg/ml–0.9 ng/ml) is also compatible with the physiologic concentrations of the proinflammatory cytokines generally found in an inflammatory site. It is concluded that MCP-1 is produced by Leydig cells and peritubular cells and that it could be involved in the mobilization and migration of leukocytes observed during testicular inflammation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages and lymphocytes are found within the testes of mammals, where they intervene in the normal testicular physiology [1]. For example, they are believed to be involved in immunoregulation and in the local regulation of normal Leydig cell development and steroidogenesis [2, 3]. They have also been shown to be involved in testicular physiopathology. In some immune activation processes such as testis inflammation (orchitis), induced by infectious agents, environmental toxins, trauma, or autoimmune reactions [1], leukocytes possess the ability to migrate from testicular blood vessels to the interstitial compartment [4, 5]. They may also migrate further to the seminiferous tubules, where they have been observed phagocytosing germ cells and Sertoli cells [6]. The damage caused in these circumstances can lead to transitory or even permanent sterility in animals and men.

Over the past ten years, the molecules responsible for the attraction of the leukocytes to an inflammatory site have been characterized and designated as members of the chemokine family. This family can be divided into 4 subfamilies (C-, CC-, CXC-, and CX3C-chemokines) depending on the relative position of the two first conserved cysteines [7]. Macrophage inflammatory protein (MIP)-1{alpha}, MIP-1ß, and monocyte chemoattractant protein-1 (MCP-1) belong to the CC-chemokine subfamily. These 3 chemokines are involved in the chemoattraction of monocytes, macrophages, and lymphocytes in various tissues, as well as in histamine release by basophils and proliferation of hematopoietic stem cells [7]. Numerous cell types including endothelial cells [8, 9], fibroblasts [9, 10], and epithelial cells [11] have been found to express MIP-1{alpha}, MIP-1ß, and MCP-1 when exposed to appropriate stimuli. Among the inflammatory cytokines able to induce MIP-1{alpha}, MIP-1ß, and MCP-1 production are bacterial lipopolysaccharide (LPS), interleukin (IL)-1{alpha}, IL-1ß, interferon-{gamma} (IFN-{gamma}), and tumor necrosis factor {alpha} (TNF-{alpha}) [7]. As the cytokines are all expressed within the testis [3, 12], the aim of this study was to investigate whether or not testicular cells can produce relevant CC-chemokines such as the macrophage chemoattractant factors MIP-1{alpha}, MIP-1ß, and MCP-1 that may be involved in the regulation of inflammatory cell influx.


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

Male Sprague-Dawley rats were purchased from Elevage Janvier (Le Genest Saint Isle, France). Rat recombinant MCP-1 was obtained from Pharmingen (Pharmingen-Becton Dickinson, le Pont de Claix, France). Enzymes for the cell preparations were purchased from Sigma (Saint Quentin Fallavier, France). All experimental and surgical procedures involving animals were approved by Veterinary Office, Ministry of Agriculture, France.

Preparation and Culture of Sertoli Cells

The isolation and culture of Sertoli cells from 20-day-old rats was carried out as described by Toebosch et al. [13] and Pineau et al. [14]. The cells were seeded at 1.5 x 106 cells/ml in Ham's F-12/Dulbecco's modified Eagle's medium (DMEM) (v:v; Life Technologies, Cergy Pontoise, France) supplemented with insulin (10 µg/ml), transferrin (5 µg/ml), gentamycin (50 µg/ml) (Life Technologies), and 10% fetal calf serum (FCS) (Costar; Polylabo, Strasbourg, France) and incubated at 32°C in a humidified atmosphere with 5% CO2 and 95% air. On Day 7 of culture, Sertoli cells contaminated with less than 2% germ cells and peritubular cells were either used as controls or exposed to various agents.

Preparation and Culture of Peritubular Cells

Peritubular cells were isolated from 20-day-old rats, according to the method described by Skinner and Fritz [15]. The cells were cultured at 32°C in Ham's F-12/DMEM supplemented with 10% FCS and became confluent after 7 days of culture. After only 3 days in culture, with daily changes of medium, purity of these cells was about 96% as assessed by the alkaline phosphatase method [16]. Furthermore, no contamination by Leydig cells or macrophages could be detected using a 3ß-hydroxysteroid dehydrogenase staining technique [17] and by immunocytochemistry using an ED2 antibody detecting an antigen specific for macrophages (Serotec-Argene, Varilhes, France). These peritubular cells were then passaged once, and at confluence they were washed with culture medium and either used as controls or exposed to various agents.

Preparation and Culture of Leydig Cells

Cell suspensions highly enriched for Leydig cells were prepared from adult testes of 90-day-old rats according to the multistep isolation method of Klinefelter et al. [18]. This procedure involves the use of testicular perfusion, enzymatic dissociation, centrifugal elutriation, and density Percoll (Pharmacia and Upjohn, Kalamazoo, MI) gradient centrifugation. After centrifugation, the Percoll gradient was divided into a fraction lighter than 1.068 g/ml that contained germ cells, macrophages, and damaged Leydig cells, and a fraction heavier than 1.068 g/ml that contained intact and steroidogenically active Leydig cells. At this stage, the purity of the Leydig cells was 94% or more, as assessed by 3ß-hydroxysteroid dehydrogenase staining of the cells. Contaminants were mainly testicular macrophages (< 4%), peritubular cells (~0.5%), and very few Sertoli and germ cells. Rat Leydig cells were cultured for 24 h in Ham's F-12/DMEM (v:v) supplemented with gentamycin (50 µg/ml), 0.1% BSA (Biosepra, Villeneuve la Garenne, France), and 10% FCS. After 12 h of culture, the cells were either used as controls or exposed to various agents.

Preparation of Spermatogonia

The isolation and culture of spermatogonia from 9-day-old rats were carried out as described by Bellvé et al. [19], with the minor modifications introduced by Dym et al. [20]. This procedure involved the use of enzymatic dissociation and then a filtration through 80- and 40-µm nylon mesh. Cells from the dissociated seminiferous tubules were separated by sedimentation velocity at unit gravity at 4°C using a 2–4% bovine BSA gradient in Ham's F-12/DMEM. The cell suspension was bottom-loaded into an SP-120 chamber in 30 ml of Ham's F-12/DMEM containing 0.5% BSA, and a gradient was simultaneously generated using 275 ml each of medium supplemented with 2% and 4% BSA, respectively. The cells were allowed to sediment for a standard period of 2.5 h, and 300 ml was then collected from the bottom of the gradient and centrifuged at 100 x g for 10 min. Pellet cells were then resuspended in Ham's F-12/DMEM supplemented with gentamycin (50 µg/ml) and 10% FCS and incubated at a density of 2.5 106/ml in a humidified atmosphere of 5% CO2, 95% air. After 2 h of culture, contaminant cells were plated, and nonadherent spermatogonia were used for RNA extraction.

Preparation of Pachytene Spermatocytes and Early Spermatids

Postmitotic germ cell preparations were obtained from testes of 90-day-old rats by mechanical dissociation [21]. These cells were separated by centrifugal elutriation into 2 populations: primary spermatocytes and early spermatids. Flow rate and/or rotor speed were changed progressively as described by Pineau et al. [21]. Cell viability was evaluated by the trypan blue exclusion test and was found to be at least 95%. Pachytene spermatocytes and early spermatids fractions were found to be about 90% pure [21]. The enriched fractions were used for RNA extraction.

Preparation and Culture of Germ Cells

The total germ cell fraction was obtained from 90-day-old rats according to the method described by Meistrich et al. [22]. Briefly, testes were trypsin digested, and the tubules were mechanically dispersed. Spermatozoa were excluded by filtration over glass wool. Total germ cells were incubated at 32°C at 10 x 106 cells/ml in PBS buffer supplemented with 6 mM lactate, 5.6 mM glucose, 0.4% BSA, and 50 µg/ml gentamycin; then total germ cells were either used as controls or exposed to various agents. The poor viability of germ cells in culture does not allow their culture for periods longer than 8 h.

Preparation and Culture of Peritoneal Macrophages

To induce a local inflammation, adult rats were injected with 35 ml (29.8 g/L) of thioglycollate medium (Sigma) into the peritoneum as described by Van Furth et al. [23]. Four days later, exudate cells were harvested by several washes with PBS. The peritoneal cells were then washed and seeded at a density of 106 cells/ml in Ham's F-12-DMEM supplemented with 50 µg/ml gentamycin and 10% FCS. One hour after incubation at 37°C, nonadherent cells were removed by repeated washes with medium.

Stimulation of Cultures

To determine whether testicular cells are able to express chemokines, confluent Sertoli cells, Leydig cells, peritubular cells, and total germ cells were incubated for 8 and 24 h with either 100 U/ml recombinant rat IFN-{gamma} (Biosource-Clinisciences, Montrouge, France), 100 U/ml recombinant rat IFN-{alpha} (Biosource), 20 ng/ml recombinant human TNF-{alpha} (R & D Systems, Abingdon, UK), 1 µg/ml Salmonella typhimurium LPS (Sigma), 1 ng/ml recombinant human IL-1{alpha} (R & D Systems), 10 ng/ml recombinant human IL-6 (R & D Systems), or 1 ng/ml recombinant human IL-1ß (R & D Systems). Furthermore, the dose-dependent protein production of MCP-1 was determined after a 24-h incubation period with 0.01 pg/ml-10 ng/ml of TNF-{alpha}, 0.01–3000 ng/ml of LPS, or 0.03 pg/ml–3 ng/ml of IL-1ß.

Northern Blotting and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Cells were harvested and pellets were stored at -20°C until RNA extraction. Total RNA was isolated from Sertoli cells, peritubular cells, Leydig cells, and total germ cells using the RNeasy kit (Qiagen, Les Ulis, France). For Northern blotting, total RNA samples (10 µg/lane) were fractionated on a 1% agarose gel and transferred to Hybond N membranes (Amersham, Les Ulis, France) as described by Sambrook et al. [24]. Probes were [{alpha}-32P]-labeled by random priming [25] and used for blot hybridization.

For RT-PCR, total RNA samples were treated with 5 U/2 µg RNA of RNAse Free Dnase I (Promega, Charbonnières, France) in the presence of 40 U/µl RNasin, an RNase inhibitor (Promega), for 30 min at 37°C. RT was performed using the superscript preamplification system kit obtained from Gibco (Life Technologies, Gaithersburg, MD). PCRs were performed on cDNA samples, using as forward primers 5'-TTCTCTGCACCATGGCGC-3', 5'-TCCTCCTGCTTGTGGCCG-3', 5'-CAGGTCTCTGTCACGCTTCT-3', and 5'-ATGGATGACATCGATGCTGC-3' and as reverse primers 5'-CTCTTTGGGGTCAGCGCA-3', 5'-CATTGACCCAGGGCTCGC-3', 5'-AGTATTCATGGAAGGGAATAG-3', and 5'-GCTGGAAGGTGGACAGTGAG-3' for MIP-1{alpha} (GenBank, accession U06435), MIP-1ß (GenBank, accession U06434), MCP-1 (GenBank, accession M57441), and ß-actin (GenBank, accession J00691) transcripts, respectively. PCR conditions were 93°C for 3 min; 93°C for 1 min, 56°C, 57°C, 54°C, or 54°C (for the transcripts, respectively) for 1 min, 72°C for 2 min, 35 cycles. Amplified products (203-base pair [bp] product for MIP-1{alpha}, 222-bp product for MIP-1ß, 525-bp product for MCP-1, and 1020-bp product for ß-actin) were analyzed electrophoretically on 1% or 2% agarose gels containing ethidium bromide. The nucleotide sequence of each PCR product was validated on both strands using the dye nucleotide cycle sequencing technique and a 373A DNA sequencer from Applied Biosystems (Foster City, CA).

Sandwich ELISA for Rat MCP-1

All the supernatants of cultured cells were stored at -80°C. A sandwich ELISA for rat MCP-1 was developed with two monoclonal antibodies as recommended by Pharmingen. Maxisorb plates (Nunc; Polylabo) were coated with 400 ng/well mouse monoclonal anti-rat MCP-1 antibody (Pharmingen) in 0.1 M Na2HPO4, pH 9, overnight at 4°C. The plates were then washed 4 times with PBS containing 0.05% Tween 20 (wash buffer). A blocking step was performed with PBS containing 1% BSA (blocking buffer) for 30 min at room temperature; the plates were then washed 3 times. To obtain linear curves, recombinant rat MCP-1 protein (Pharmingen) prepared in Ham's F-12/DMEM supplemented with 10% FCS ranging from 5 to 4000 pg/ml was included in each ELISA plate. Various dilutions of cell culture supernatants were added. After 90 min at 37°C, the plates were washed 4 times, and 100 ng/well biotinylated mouse monoclonal anti-rat MCP-1 antibody (Pharmingen) in blocking buffer containing 0.05% Tween 20 was added. After 1 h at 37°C, plates were washed 4 times, and each well received 100 µl streptavidin-horseradish peroxidase-conjugated (Dako, Trappes, France; 1/5000 in blocking buffer containing 0.05% Tween 20) for 30 min at room temperature. After 4 washes, 100 µl substrate solution (o-phenylenediamine dihydrochloride) (Sigma) was added. The reaction was stopped by addition of 150 µl/well HCl 1N, and absorbance was read at 492 nm on an ELISA reader. MCP-1 concentrations were calculated by interpolation from the standard curve.

DNA Measurement

DNA was quantified fluorometrically with Hoechst 33258 reagent (Sigma) using calf thymus DNA as the standard [26]. Each cell sample was homogenized in phosphate-saline buffer (2 M NaCl, 2 mM EDTA, 30 mM Na2HPO4, 20 mM NaH2PO4, pH 7.4) and sonicated briefly. The Hoechst 33258 reagent was added to 3.6 µg/ml final concentration, and fluorescence measurements were performed on a fluorocolorimeter (excitation filter 365 nm; emission filter 450 nm). Triplicate dilutions in PBS of calf thymus DNA ranging from 1.56 to 50 µg/ml were included to determine DNA concentrations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MIP-1{alpha}, MIP-1ß, and MCP-1 mRNA Expression in Sertoli Cells, Germ Cells, Peritubular Cells, and Leydig Cells

To determine expression of MIP-1{alpha}, MIP-1ß, and MCP-1, RT-PCR amplifications were performed from samples prepared from Sertoli cells, spermatogonia, pachytene spermatocytes, early spermatids, peritubular cells, and Leydig cells cultured in the absence or presence of inflammatory cytokines or of bacterial LPS. MIP-1{alpha} and MIP-1ß RNAs were not detected in Sertoli cell, germ cell, peritubular cell, or Leydig cell preparations whether they were exposed or not to the various stimuli, but were present in the peritoneal macrophages used as a positive control (Fig. 1). MCP-1 RT-PCR product was consistently found both in peritoneal macrophages and in Sertoli cells stimulated with TNF-{alpha} and IL-1ß and to a lesser extent with IL-1{alpha} and LPS. The MCP-1 product was also found in peritubular cells and Leydig cells exposed or not to the various stimuli (Fig. 1). In contrast, the MCP-1 RNA was not detected in highly enriched populations of spermatogonia, pachytene spermatocytes, and early spermatids.



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  		FIG. 1. MIP-1{alpha}, MIP-1ß, and MCP-1 mRNA expression in Sertoli, germ, peritubular, and Leydig cells. RT-PCR amplification of MIP-1{alpha}, MIP-1ß, and MCP-1 was performed on cDNA samples from peritoneal macrophages (Perit. Macro.) used as positive control, as well as from control (Cont.) Sertoli cells, spermatogonia (Gonia), pachytene spermatocytes (SP), early spermatids (ES), Leydig cells, and peritubular cells, and from the same cells (except for the germ cells) exposed for 8 h to 100 U/ml IFN-{alpha}, 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha}, 1 µg/ml LPS, 1 ng/ml IL-1{alpha}, 10 ng/ml IL-6, and 1 ng/ml IL-1ß. MIP-1{alpha}, MIP-1ß, and MCP-1 primers amplified a 203-bp, 222-bp, and 525-bp product, respectively. ß-Actin amplification was used as positive control for PCR and RNA preparation. The expected 1020-bp product was detected for all cellular preparations. The results shown are representative of two separate experiments

Northern Blot Analysis of MCP-1 in Peritubular Cells, Sertoli Cells, and Germ Cells

To evaluate the level of expression of MCP-1 mRNA, peritubular cells, Sertoli cells, and total germ cells were stimulated with different proinflammatory cytokines prior to extraction of mRNA. Northern blot analysis was then performed using an MCP-1 probe. While high levels of MCP-1 transcripts of 1.3 kilobases (kb) and 1 kb were detected in peritubular cells exposed to TNF-{alpha}, LPS, IL-1{alpha}, and IL-1ß, there were lower levels in the cells exposed to IFN-{gamma} and very low levels in cells incubated with IFN-{alpha} and IL-6 and in unexposed cells (Fig. 2A). In contrast, the MCP-1 transcripts were not detected in Sertoli cells or in germ cells using the Northern blot technique (Fig. 2, B and C).



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  		FIG. 2. Northern blot analysis of MCP-1. Peritubular cells (A), Sertoli cells (B), and total germ cells (C) were cultured for 8 h in absence (Cont.) or in presence of 100 U/ml IFN-{alpha}, 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha}, 1 µg/ml LPS, 1 ng/ml IL-1{alpha}, 10 ng/ml IL-6, and 1 ng/ml IL-1ß. Total RNAs were isolated, loaded on agarose gels (10 µg RNA per lane), and fractionated. The blots were hybridized with 32P-labeled MCP-1 probe and autoradiographed. The quality of RNA preparation and equal loading were assessed by hybridization with a ß-actin probe. The results shown are representative of two separate experiments

In order to investigate the ontogenesis of MCP-1 mRNA expression, Northern blot analysis was performed on peritubular cell mRNA preparations from 9-day-old (immature), 20-day-old (prepubertal), and 45-day-old (pubertal) rats (Fig. 3). Two MCP-1 mRNAs were found in peritubular cells at all ages, either in the absence of stimuli (weak signals) or in the presence of IFN-{gamma} (strong signals), TNF-{alpha}, and LPS (very strong signals).



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  		FIG. 3. Northern blot analysis of MCP-1 in peritubular cells prepared from 9-, 20-, and 45-day-old rats. Peritubular cells were incubated in absence (Cont.) or presence of 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha}, and 1 µg/ml LPS for 16 h. Total RNAs were isolated, loaded on agarose gels (10 µg RNA per lane), and fractionated. The blots were hybridized with 32P-labeled MCP-1 probe and autoradiographed. The quality of RNA preparation and equal loading were assessed by hybridization with a ß-actin probe (not shown). The results shown are representative of two separate experiments

Production of MCP-1 by Peritubular Cells and Leydig Cells

Peritubular cells, Sertoli cells, total germ cells, and Leydig cells were cultured for 24 h in the presence of 100 U/ml IFN-{alpha}, 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha}, 1 µg/ml LPS, 1 ng/ml IL-1{alpha}, 10 ng/ml IL-6, 1 ng/ml IL-1ß, or medium alone; and rat MCP-1 was measured in the culture media using a sandwich ELISA. MCP-1 was not detected in the media prepared from Sertoli cell and germ cell cultures, whether or not these cells were exposed to the various inducing factors (data not shown). In contrast, a low constitutive MCP-1 production by peritubular cells and Leydig cells was observed (Fig. 4). Furthermore, a significant stimulation of peritubular cell MCP-1 was seen when these cells were stimulated with IFN-{alpha}, IFN-{gamma}, TNF-{alpha}, LPS, IL-1{alpha}, and IL-1ß. In particular, a 7- to 10-fold stimulation was observed with LPS, TNF-{alpha}, IL-1{alpha}, and IL-1ß. In Leydig cells, of all the factors tested, only IL-1ß was able to significantly stimulate MCP-1 production.



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  		FIG. 4. Production of MCP-1 by peritubular cells and Leydig cells. Peritubular cells and Leydig cells in primary culture were incubated in absence (Cont.) or in presence of 100 U/ml IFN-{alpha}, 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha}, 1 µg/ml LPS, 1 ng/ml IL-1{alpha}, 10 ng/ml IL-6, and 1 ng/ml IL-1ß for 24 h. Culture media were harvested and MCP-1 production was quantified using a specific ELISA described in Materials and Methods. The data represented are a mean ± SD of 3 wells each assayed in duplicate and are representative of two separate experiments. After Student's t-test, differences considered statistically significant in relation to controls were noted; *P < 0.05, **P < 0.01, and ***P < 0.005

Kinetics of Peritubular Cell MCP-1 mRNA Expression and MCP-1 Production

In order to investigate the kinetics of MCP-1 mRNA and protein expression, peritubular cells were cultured in the absence or presence of LPS, TNF-{alpha}, or IL-1ß from 0 to 72 h. Peritubular cell MCP-1 mRNAs were markedly stimulated as early as 2 h following exposure to LPS, TNF-{alpha}, and IL-1ß; and a maximal expression was observed at 8 h for LPS and TNF-{alpha}, and at 16 h for IL-1ß, before declining after 8 h for LPS and TNF-{alpha} and after 24 h for IL-1ß (Fig. 5). While the 1-kb form was not detected after only 1 or 2 h of culture, the two MCP-1 transcripts were consistently observed after 4 h (Fig. 5). LPS, TNF-{alpha}, and IL-1ß were all found to stimulate this peritubular cell MCP-1 production significantly (P < 0.005) after 8 h of exposure (Fig. 6). Maximal levels were reached from 24 h for LPS and 48 h with TNF-{alpha} and IL-1ß.



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  		FIG. 5. Kinetics of peritubular cell MCP-1 mRNA expression after exposure to LPS, TNF-{alpha}, and IL-1ß. Peritubular cells were cultured in presence of 1 µg/ml LPS, 0.1 ng/ml TNF-{alpha}, and 0.1 ng/ml IL-1ß for 1, 2, 4, 8, 16, 24, 48, and 72 h. Total RNAs were isolated, loaded on agarose gels (10 µg RNA per lane), and fractionated. The blots were hybridized with 32P-labeled MCP-1 probe and autoradiographed. The quality of RNA preparation and equal loading were assessed by hybridization with a ß-actin probe. The results represent MCP-1 mRNA autoradiograms and graphs showing the MCP-1 1.3-kb and 1-kb mRNA/ß-actin mRNA expression ratio in relative density to correct for mRNA loading differences and indicate the quantity of MCP-1 mRNA expression. These results are representative of two separate experiments



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  		FIG. 6. Kinetics of MCP-1 production by peritubular cells in culture. Peritubular cells were cultured in absence (squares) or presence of 30 ng/ml LPS (solid circles), 0.1 ng/ml TNF-{alpha} (open circles), or 0.1 ng/ml IL-1ß (inverted triangles) for 0–72 h. MCP-1 production was quantified in culture media by ELISA. The data presented are mean ± SEM for 3 wells, each assayed in duplicate, and are representative of two separate experiments

Effect of Increasing Concentrations of LPS, TNF-{alpha}, and IL-1ß on Peritubular Cell MCP-1 Production

When increasing concentrations of LPS, TNF-{alpha}, and IL-1ß were added to peritubular cells, a dose-dependent production of MCP-1 occurred (Fig. 7). The half-maximal response (ED50) for MCP-1 production was obtained in constructing the Eadie-Hofstee plots where the ED50 is represented by the slope of linear regression (not shown). The ED50 occurred at 0.9 ng/ml for LPS, at 0.01 ng/ml for TNF-{alpha}, and at 0.002 ng/ml for IL-1ß (Fig. 7). The minimal concentrations generating a significant MCP-1 production (P < 0.005) were >= 1 ng/ml for LPS, >= 0.03 ng/ml for TNF-{alpha}, and >= 0.01 ng/ml for IL-1ß.



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  		FIG. 7. Effects of LPS, TNF-{alpha}, and IL-1ß on MCP-1 production by peritubular cells in culture. Peritubular cells were cultured with 1 pg/ml to 3 µg/ml of LPS, 0.01 pg/ml to 3 ng/ml of TNF-{alpha}, and 0.03 pg/ml to 3 ng/ml of IL-1ß for 24 h. MCP-1 was measured in culture media using a specific ELISA, as described in Materials and Methods. The data presented are mean ± SEM for 3 wells, each assayed in duplicate, and are representative of two separate experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An influx of circulating monocytes, lymphocytes, and neutrophils has been reported in testicular inflammation [1]. However, the molecular mechanisms underlaying these infiltrations that can lead to sterility [1] have virtually not been studied yet. We hypothesize that some testicular cell subpopulations produce chemokines that may be active in specific mechanisms important for leukocyte attraction.

To the best of our knowledge, Hakovirta et al. [27] were the first to study a chemokine within the testis. Using an immunohistochemical technique with an anti-human MIP-1{alpha} antibody developed by Dr. S. Wolpe (Genetics Institute, Cambridge, MA), these authors claimed that MIP-1{alpha} is present in both germ cells and Leydig cells. We could not repeat this experiment, as Dr. S. Wolpe has run out of this antibody. Therefore, we used a commercial anti-recombinant rat MIP-1{alpha} (Pepro Tech-TEBU, Le Perray-en-Yvelines, France) to perform immunohistochemistry and found that the only specific signal observed was located in the endothelial cells of blood testicular vessels (data not shown). This result is in agreement with the detection of MIP-1{alpha} in human blood vessel endothelial cells as already described by Adams et al. [28]. This is also consistent with our RT-PCR study indicating that MIP-1{alpha} transcripts could not be detected in Sertoli, germ, peritubular, or Leydig cells. The reason for this discrepancy between the data of Hakovirta et al. [27] and our own data is unclear, but it is likely that it results from the fact that the antibody used by the former was an anti-peptide and anti-human, which by nature is susceptible to being less specific than an anti-recombinant rat protein, the one used by us.

In contrast to what was observed for MIP-1{alpha}, our approach based on the primary culture of different populations of testicular cells demonstrates the ability of Leydig cells and peritubular cells to express and produce MCP-1 mRNA and protein. While MCP-1 transcripts were detected by RT-PCR in Sertoli cells, Northern blot analysis and ELISA (data not shown) did not substantiate this finding. This may indicate that there exists a very low MCP-1 expression rate in these cells that is detectable only by the very sensitive PCR technique. However, it is at least equally probable that the positive signals observed result from very low contamination of interstitial fibroblasts or of other MCP-1-expressing cells. MCP-1 transcripts were also detected in highly enriched population of testicular macrophages (data not shown); however, the rarity of this material prevented further study of this cell category.

The expression of MCP-1 mRNA and protein in rat peritubular cells is strongly stimulated by LPS, TNF-{alpha}, and the cytokines IFN-{gamma}, IL-1{alpha}, IL-1ß, which are remarkably potent stimulators. High levels of IL-1 have been observed in both rat and human testis and testicular cells [29, 30]. Sertoli cells constitute a major source of IL-1{alpha}, and this production increases with testicular development [31, 32]. Isolated germ cells also produce IL-1{alpha} [30, 33]. In contrast, Leydig cells essentially secrete only IL-1ß [30, 34]. IL-1 type I and type II receptors have been shown to be expressed in Sertoli cells, germ cells, peritubular cells, Leydig cells, and resident macrophages, which argues for multicellular targets of IL-1 within the testis [35–37]. The fact that peritubular and Leydig cells produce MCP-1 after stimulation by IL-1 is a new demonstration of the presence of IL-1 receptors on peritubular and Leydig cell plasmic membranes and strongly suggests the possibility that IL-1 (IL-1ß in particular) is involved in the pathological testicular immune response.

The presence of TNF-{alpha} and its receptors has also been demonstrated in the testis [3, 12, 38], and more specifically in Leydig and Sertoli cells [38, 39]. TNF-{alpha} is secreted by macrophages in the interstitial tissue and by germ cells in the seminiferous tubule [40]. As TNF-{alpha} also appears to be a very potent activator of MCP-1, we hypothesize that in an inflammatory situation, TNF-{alpha} secreted by activated interstitial macrophages or by germ cells could trigger MCP-1 production by peritubular cells. This, however, requires experimental demonstration.

Type I IFNs ({alpha}/ß) and type II IFN ({gamma}) are also expressed within the testis. While IFN ({alpha}/ß) is produced by most testicular cell types, IFN-{gamma} production is restricted to early spermatids and Leydig cells [41, 42]. In this paper, we show that peritubular cells are able to produce MCP-1 when stimulated by IFN-{gamma} but not by type I IFNs. It seems more than likely that it is the Leydig cell IFN-{gamma} that is involved in peritubular cell MCP-1 regulation during inflammation, since Leydig cells are located in the vicinity of peritubular cells.

In contrast to the other cytokines tested here for their ability to induce MCP-1, IL-6, which is known to be produced by Sertoli cells [43, 44], is the only cytokine we have tested that is unable to induce MCP-1. While our results are similar to those of Sica et al. [9] in showing that IL-1, TNF-{alpha}, and LPS (but not IL-6) induce MCP-1 gene expression in human endothelial cells, they contrast with those of Biswas et al. [45], who demonstrated that IL-6 stimulates MCP-1 expression in peripheral blood mononuclear cells and in the U937 cell line. Our data show the presence of 2 different MCP-1 transcripts of 1 and 1.3 kb in the testicular cell culture stimulated for more than 2 h. Two MCP-1 mRNAs have also been observed in human vascular endothelial cells [46] while only one was detected in rat spleen cells [10]. These different experimental results obtained from different organs may reflect organ/tissue differences in MCP-1 mRNA processing.

MCP-1 expression by peritubular cells stimulated by IFN-{gamma}, LPS, and TNF-{alpha} was observed in immature as well as prepubertal and pubertal rats. These results indicate that the peritubular cell MCP-1 system is operational very early during testicular development.

The ED50 of MCP-1 production by peritubular cells found here for TNF-{alpha} (0.01 ng/ml), IL-1ß (2 pg/ml), and LPS (0.9 ng/ml) are compatible with the concentrations of cytokines usually found in an inflammatory site and with the effects of these factors in other biological systems. Indeed, Matthews and Neale [47] have reported that the ED50 of TNF-{alpha}, measured in a cytotoxicity assay using the TNF-susceptible murine L-929 cell line, was about 0.02 ng/ml. In the same way, the ED50 of IL-1ß measured in a cell proliferation assay using the murine helper T cell line was about 5 pg/ml [48]. The LPS effects on MCP-1 production with ED50 of about 1 ng/ml seem to indicate a CD14-dependent signaling pathway [49]; however, CD14 receptors have not yet been characterized in peritubular cells.

MCP-1 mRNAs and protein expression were stimulated in peritubular cell cultures as early as 2 and 8 h post-LPS and -IL-1ß addition, respectively. These rapid kinetics are in total agreement with those observed for MCP-1 production in human endothelial cells [8], human eosinophils [50], rat type II alveolar epithelial cells [51], and rat pulmonary alveolar macrophages [52]. We show that the kinetics of MCP-1 protein production are compatible with a rapid mobilization of the peritubular cells in an inflammatory process.

Leydig cells represent the second cell type able to produce MCP-1 in testis. While the amount of MCP-1 that can be produced by Leydig cells is similar to that produced by peritubular cells, only IL-1ß appeared to be able to stimulate Leydig cell MCP-1 production. As testicular IL-1ß is produced by macrophages and by Leydig cells themselves, it is probable that Leydig cell MCP-1 secretion is essentially under paracrine (macrophages) and/or autocrine (Leydig cells) control.

In conclusion, the high levels of expression and production of MCP-1 mRNA and protein by peritubular and Leydig cells, the high sensitivity of the peritubular cell response to a number of inflammatory cytokines, and the rapid response are together strong arguments in support of the hypothesis that Leydig cells and peritubular cells constitute the key factors in the mechanism of circulating leukocyte mobilization and migration from blood to the testis in the case of testicular inflammation. The particular topographic localization of Leydig cells at the interface of the blood vessels and interstitial compartment, and of peritubular cells at the interface of the interstitial compartment and seminiferous tubules, strongly reinforces this hypothesis. Therefore, we hypothesize that the Leydig cells may be involved in the chemoattraction of leukocytes from blood vessels to the interstitial compartment, while the peritubular cells may be responsible for leukocyte migration from the interstitial compartment to the seminiferous tubules.


   FOOTNOTES
 
First decision: 5 November 1999.

1 This work has been supported by INSERM, the Ministère de l'Education Nationale de la Recherche et de la Technologie, the Fondation pour la Recherche médicale (FRM), the Association pour la Recherche sur le Cancer (ARC), the Ligue nationale contre le cancer, the Région Bretagne, and the Fondation Langlois. Back

2 Correspondence: Michel Samson, Université de Rennes I, GERM-INSERM U. 435, Campus de Beaulieu, 35042 Rennes cedex, Bretagne, France. FAX: 33 2 99 28 16 13; michel.samson{at}rennes.inserm.fr Back

Accepted: December 31, 1999.

Received: October 1, 1999.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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