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REPRODUCTION-DEVELOPMENT |
-Inducible Protein-10 Is Induced by the Sendai Virus in Human and Rat Testicular Cells
GERM-INSERM, U-435, Université de Rennes I, Campus de Beaulieu (R.L.G., T.M., F.A., B.J., M.S.), 35042 Rennes, Bretagne, France; Service d’Urologie, CHU Ponchaillou (J.-J.P.), 35000 Rennes, Bretagne, France; and Service de Bactériologie-Virologie, CHU Ponchaillou (A.R.), 35000 Rennes, Bretagne, France
Address all correspondence and requests for reprints to: Dr. Michel Samson, Université de Rennes I, GERM-INSERM, U-435, Campus de Beaulieu, 35042 Rennes Cedex, Bretagne, France. E-mail: .
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Abstract |
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, and interferon-
-inducible protein-10. In rat peritubular cells exposed to the Sendai virus, MCP-1 production was time and dose dependent. In contrast, rat germ cells did not produce these chemokines. Chemokine synthesis was detected in human Leydig cells exposed to the Sendai virus, but not in human total germ cells, suggesting that rats and humans display similar responses in terms of chemokine production. MCP-1, regulated on activation normal T cell expressed and secreted protein, growth-related oncogene-, and interferon--inducible protein-10 have been reported to be chemoattractants for a large variety of leukocytes. The ability of the Sendai virus to induce chemokine production in somatic cells (mostly peritubular and Leydig cells) may therefore increase the recruitment of leukocytes to sites of infection. |
Introduction |
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Chemokines are known to regulate the recruitment and functional activation of leukocytes, and the production of some chemokines has been reported to increase in human and animal cells during viral infections (4). Human cytomegalovirus induces the production of regulated on activation normal T cell expressed and secreted protein (RANTES) in cultured human fibroblasts (5), whereas EBV triggers the production of IL-8 and macrophage inflammatory protein-1 (MIP-1) in cultured human neutrophils (6). HIV has also been shown to increase the production of RANTES, MIP-1, MIP-1ß, and IL-8 mRNA in myeloid cells (7), and Newcastle disease virus stimulates the synthesis of interferon- (IFN)-inducible protein-10 (IP-10) in U251, a human astrocytoma cell line, and in rat astrocytes (8). Apart from data indicating an increase in IL-8 levels in the seminal plasma of men infected with mumps virus and presenting orchitis (9), nothing is known about the relationship between infection and chemokine production in the genital tract. More specifically, no data are available concerning the mechanisms underlying leukocyte infiltration during viral orchitis and the possible involvement of virally induced chemokines.
In a series of recent studies we and others have demonstrated that rat testicular cells in primary culture produce chemokines in response to various proinflammatory cytokines. Thus, monocyte chemotactic protein-1 (MCP-1), IP-10, and growth-related oncogene (GRO) are produced in large quantities by Leydig cells, peritubular cells, and, under certain conditions, Sertoli cells (10, 11, 12).
In this study we investigated the production of chemokines, RANTES, MCP-1, GRO, and IP-10 in primary cultures of rat testicular cells and human Leydig cells infected with the Sendai virus, a virus similar to the mumps virus that displays tropism toward testicular cells (13, 14, 15, 16).
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Materials and Methods |
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antibody (500-p74; lot 017E571) was obtained from PeproTech, Inc. (London, UK), and monoclonal antihuman IP-10 antibody (MAB266; lot ADN02) was purchased from R\|[amp ]\|D Systems, Inc. (Abingdon, UK).
Rat Sertoli cell preparation
Sertoli cells were isolated from 20-d-old rats as previously described (17). The Sertoli cell preparations, containing 2% contaminating germ cells and less than 2% peritubular cells, were seeded in Ham’s F-12/DMEM (vol/vol) (Life Technologies, Inc., Cergy Pontoise, France) at a density of 1.0 x 105 cells/ml for immunocytochemistry or at a density of 1.5 x 106 cells/ml in Ham’s F-12/DMEM supplemented with 10 µg/ml insulin (Sigma, Saint Quentin-Fallavier, France), 5 µg/ml transferrin (Sigma), 50 µg/ml gentamicin (Life Technologies, Inc.), and 5% FCS (Costar, Brumath, France). The cells were incubated at 32 C in a humidified atmosphere consisting of 5% CO2-95% air. On the second day of culture, germ cell contaminants were removed by brief washing with PBS. On d 4 of culture, some of the cells were removed for use as controls (incubated with allantoic liquid), and the remaining cells were treated with the Sendai virus. Supernatants were collected for ELISA, and the cells were suspended in guanidium thiocyanate for RNA extraction or were fixed with formaldehyde for immunocytochemistry.
Rat peritubular cell preparation
Peritubular cells were isolated from 20-d-old rats according to a published method (18). The cells were cultured at 32 C in Ham’s F-12/DMEM supplemented with 5% FCS and became confluent after 6–8 d of culture. They were then passaged twice and seeded at a density of 1.5 x 106 cells/ml (or at a density of 0.5 x 105 cells/ml for immunocytochemistry). At confluence, the cells were washed with PBS and treated as described above for rat Sertoli cells.
Rat testicular macrophages and Leydig cell preparation
Cell suspensions highly enriched in Leydig cells and testicular macrophages were prepared from adult rat testes as previously described (19). This procedure involved testicular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll density gradient centrifugation. After centrifugation, the Percoll gradient was divided into a fraction with a density below 1.068 g/ml, which contained germ cells, macrophages, and damaged Leydig cells, and a fraction with a density greater than 1.068 g/ml, which contained intact and steroidogenically active Leydig cells. At this stage the Leydig cell preparation was at least 94%, as assessed by 3-hydroxysteroid dehydrogenase staining of the cells. Rat Leydig cells were cultured for 24 h in Ham’s F-12/DMEM supplemented with 50 µg/ml gentamicin, 0.1% BSA (Biosepra, Villeneuve la Garenne, France), and 10% FCS. After 1 d of culture, the cells were used for RNA extraction, and supernatants were stored at -80 C until use.
Human Leydig cell preparation
Testes were obtained from patients who underwent orchidectomy for prostate carcinoma at Pontchaillou Hospital (Rennes, France) following a procedure approved by local ethical committees. Human Leydig cells were isolated and cultured as previously described (20). Briefly, the tissue was carefully dissected with forceps, then digested for 90–120 min at 37 C in culture medium containing 1 mg/ml collagenase, 10 µg/ml DNase, and 1 µg/ml soybean trypsin inhibitor. The digested tissue was filtered through a nylon gauze, and the cells were washed by centrifugation/resuspension. The cell suspension was applied to a four-layer discontinuous Percoll gradient. The cell band corresponding to the Leydig cells in fraction of density (1.068–1.075) was collected and washed by centrifugation/resuspension. Finally, the Leydig cells were cultured in Ham’s F-12/DMEM medium supplemented with 10 µg/ml insulin (Sigma), 5 µg/ml transferrin (Sigma), 100 U/ml penicillin-streptomycin (Life Technologies, Inc.), 10 µg/ml vitamin C (Sigma), 10 µg/ml vitamin E (Sigma), 100 mIU/ml hCG (Sigma), and 2% FCS (Costar) (21).
Human total germ cells
The human total germ cell fraction was obtained from human testes as described by Meistrich et al. (22), using a method adapted from that used in the rat. Briefly, testes were treated with trypsin, and the tubules were mechanically dispersed. Spermatozoa were excluded by filtration through glass wool. The germ cells, which passed through the glass wool, were incubated at 32 C at a density of 10 x 106 cells/ml in 6 mM lactate, 5.6 mM glucose, 0.4% BSA, and 50 µg/ml gentamicin in PBS.
Rat spermatogonium preparation
Testes of 9-d-old Sprague Dawley rats were excised and decapsulated. Seminiferous epithelial cells were dispersed by trypsin treatment and separated as previously described (23). Briefly, the decapsulated testes were resuspended in PBS supplemented with collagenase (1.5 mg/ml) and deoxyribonuclease (1 µg/ml) and incubated at 32 C for 15 min in a shaker operating at 100 rpm. After two washes in PBS, small fragments of seminiferous tubules, mostly devoid of interstitial cells, were transferred to PBS supplemented with collagenase (1.5 mg/ml), hyaluronidase (1.5 mg/ml), trypsin (0.5 mg/ml), and deoxyribonuclease (1 µg/ml) and incubated for 25 min under the conditions described above. The dispersed cells were washed three times with PBS and filtered through nylon mesh, once through a mesh with 80-µm pores and once through a mesh with 40-µm pores. Cells from the dissociated seminiferous tubules were then separated by velocity sedimentation at unit gravity at 4 C using a 2–4% BSA gradient in Ham’s F-12/DMEM. The cells were bottom-loaded into an SP-120 chamber in 30 ml Ham’s F-12/DMEM supplemented with 0.5% BSA, and a gradient was immediately generated with 275 ml each of medium supplemented with 2% and 4% BSA. The cells were allowed to sediment for a standard period of 2.5 h, and 35 fractions of 15 ml were then collected. Cell fractions 16–21, corresponding to 2.8–3% BSA, were pooled and centrifuged at 100 x g for 10 min. They were then resuspended in Ham’s F-12/DMEM at a density of 2.5 x 106/ml and incubated for 2–3 h at 32 C in a humidified atmosphere consisting of 5% CO2-95% air to eliminate contaminating somatic myoid and Sertoli cells. A cell population enriched in spermatogonium cells (purity > 90%, based on morphological criteria) was then collected and incubated in Ham’s F-12/DMEM for 24 h in the presence or absence of the Sendai virus (100 hemagglutinant units/ml).
Rat pachytene spermatocyte and early spermatid preparations
Testes from 90-d-old rats were treated with trypsin and fractionated by centrifugal elutriation using a JE-6 rotor (Beckman Coulter, Inc., Palo Alto, CA). The pachytene spermatocyte and early spermatid fractions were more than 90% pure (24). These cells were cultured at 32 C at densities of 2.5 x 106 and 8 x 106 cells/ml, respectively, as previously described (24), and were treated as indicated above for Sertoli cells.
RNA extraction and Northern blot analysis
Total RNA was extracted from the various types of cell with the RNeasy mini kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. It was stored in diethylpyrocarbonate-treated water at -80 C. Total RNA was quantified by measuring absorbance at 260 nm with the usual conversion factor of 1 U at 260 nm being equivalent to 40 µg/ml. Total RNA (10 µg) was denatured in formamide-formaldehyde buffer, subjected to electrophoresis in a 2% formaldehyde-1% agarose gel, and transferred to a Hybond N membrane (Amersham Pharmacia Biotech, Les Ulis, France), to which it was cross-linked by UV irradiation (25). Probes were -32P labeled by random priming (26) and used for blot hybridization.
Sandwich ELISA for rat MCP-1 and human RANTES, IP-10, and MCP-1
The supernatants of the cultured cells were stored at -80 C. A rat MCP-1 sandwich ELISA was performed with two monoclonal antibodies (11). Human RANTES and MCP-1 were determined in supernatants with a commercially available enzyme immunoassay purchased from Cytimmune Sciences, Inc. (College Park, MD). A commercially available kit from TEBU (Le Perray-en-Yvelines, France) was used to determine IP-10, according to the manufacturer’s protocol.
DNA measurement
DNA was quantified fluorometrically with Hoechst 33258 reagent (Sigma), using calf thymus DNA as the standard (27). Each cell sample was homogenized in a PBS buffer (2 M NaCl, 2 mM EDTA, 30 mM Na2HPO4, and 20 mM NaH2PO4, pH 7.4) and sonicated briefly. Hoechst 33258 reagent was added to a final concentration of 3.6 µg/ml, and fluorescence was measured with a fluorocolorimeter (excitation filter, 365 nm; emission filter, 450 nm). Triplicate dilutions of calf thymus DNA in PBS to concentrations of 1.56–50 µg/ml were included to determine DNA concentrations.
Immunocytochemical staining
After viral infection, peritubular and Sertoli cells in primary culture were fixed by incubation in 4% paraformaldehyde and permeabilized in methanol. Immunostaining for GRO and IP-10 was performed as previously described (12).
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Results |
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, and IP-10 mRNA production in rat testicular cells exposed to the Sendai virus, and IP-10 mRNA levels was performed on samples prepared from testicular macrophages, Leydig cells, Sertoli cells, peritubular cells, spermatogonia, pachytene spermatocytes, and round spermatids, cultured in the absence or presence of the Sendai virus (Fig. 1
). We did not detect mRNA for any of the chemokines studied in germ cells. In contrast, testicular macrophages, Leydig cells, Sertoli cells, and peritubular cells contained various amounts of chemokine mRNA. In the absence of virus, no chemokine production was detected in Sertoli and peritubular cells, whereas testicular macrophages and Leydig cells produced small amounts of chemokine transcripts. In all of these cell types, chemokine production was markedly induced or stimulated (Leydig cells and testicular macrophages) by exposure to the Sendai virus. MCP-1 and GRO were synthesized principally by testicular macrophages, Leydig cells, and peritubular cells. In contrast, RANTES was produced mainly by Sertoli cells, whereas IP-10 was produced by Leydig cells and, to a lesser extent, by Sertoli and peritubular cells.
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, and IP-10 mRNA synthesis in rat peritubular cells and Sertoli cells exposed to the Sendai virus
). MCP-1, GRO, and IP-10 transcripts were detected in peritubular cells after 4 h of exposure to the virus (Fig. 2A), RANTES production was detected in peritubular cells 16 h later (Fig. 2A). In Sertoli cells, GRO and IP-10 transcripts were detected after as little as 4 h of exposure to the virus, and MCP-1 and RANTES transcripts were detected after 8 h (Fig. 2B).
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). No significant difference was measured in conditioned media of testicular macrophages in the absence or presence of virus. In contrast, MCP-1 levels in the conditioned media of peritubular cell were twice those in Leydig cell media and 7.5 times higher than those in Sertoli cell media (Fig. 3). As peritubular cells are the principal producers of MCP-1 within the testis, we used these cells for studies of the kinetics of Sendai virus-induced MCP-1 production and dose-response studies.
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). Maximal levels were reached after 72 h. The minimum concentration of virus that generated significant MCP-1 production was 5 HAU (hemagglutination units)/ml, and MCP-1 production was maximal with 100 HAU/ml (Fig. 5).
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and IP-10 in rat testicular cells exposed to the Sendai virus and IP-10 proteins were present in peritubular, Sertoli, and Leydig cells exposed to the Sendai virus, we used an immunocytochemical technique. No immunostaining was observed in peritubular cells and Sertoli cells cultured in the absence of the Sendai virus, whereas in the presence of the virus these cells displayed strong perinuclear staining with both the anti-GRO antibody (Fig. 6, B and E) and the anti-IP-10 antibody (Fig. 6, C and F). The Leydig cells displayed nonspecific staining that could not be eliminated regardless of whether the virus was present (data not shown).
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, and IP-10 mRNA in human germ cells and Leydig cells exposed to the Sendai virus, and IP-10 in a mixed preparation of human germ cells and in human Leydig cells. Chemokine synthesis was not investigated in human peritubular and Sertoli cells due to technical difficulties; we were unable to obtain sufficient quantities of these cells in sufficiently pure preparations. Northern blot analyses showed that whereas germ cells did not synthesize the studied chemokines, human Leydig cells produced MCP-1, RANTES, and IP-10 transcripts if exposed for 8 h to the Sendai virus. In contrast, GRO mRNA was not detected (Fig. 7).
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). At 72 h, IP-10 was by far the most abundant of the chemokines studied, with its levels 3.5 times and 7 times those of RANTES and MCP-1, respectively.
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Discussion |
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The Sendai virus induced the production of MCP-1, RANTES, GRO, and IP-10 transcripts after 4–8 h of exposure to virus, and the corresponding proteins were secreted into the supernatants of cell cultures after 8–16 h. This timing of chemokine production is consistent with that previously observed in Sendai virus-infected macrophages (30). In previous studies we have shown that peritubular cells produce MCP-1 and IP-10 transcripts if stimulated with IL-1ß and IFN, respectively, after only 2 h of treatment (11, 12). This suggests that the Sendai virus may rapidly and directly activate transcriptional systems such as the nuclear factor-
B system and IFN regulatory factors (29, 36, 37) involved in the up-regulation of chemokine gene expression. Alternatively, it may activate the secretion of cytokines such as IL-1ß and IFNs, inducing chemokine gene expression via autocrine regulation. Indeed, the expression of IP-10 and MCP-1 genes is induced by IFNs in a few cell types (38, 39), including Leydig cells (10), and we have shown that the production of IP-10 and MCP-1 by peritubular cells is stimulated by both IFN and IFNß (11, 12). Moreover, the Sendai virus is known to induce the production of IFN and IFN by somatic testicular cells (13, 14). Overall, these results suggest a potential role for cytokine intermediates in Sendai virus-dependent activity.
The sensitivity of peritubular cells, which produced the largest amounts of MCP-1, was evaluated with various concentrations of the Sendai virus; 5 HAU/ml was sufficient to induce significant levels of MCP-1 production, but 100 HAU/ml was the optimal concentration, leading to the production of maximal quantities of MCP-1. The Sendai virus concentration resulting in maximal IL-6 production by rat liver macrophages is 1250 HAU/ml (40), whereas 10 HAU/ml Sendai virus is sufficient to induce IFN production by peripheral blood mononuclear cells (41). Therefore, peritubular cells seem to be much more sensitive than liver macrophages or peripheral blood mononuclear cells. Although the peritubular cells are not the target cells for the Sendai virus in vivo, the high sensitivity of this cell type to the Sendai virus renders it a good in vitro model for studying cellular responses to the Sendai virus.
Testicular macrophages and Leydig cells are probably the initial testicular target of viruses circulating in the bloodstream due to their close proximity to blood vessels. Damage to the Leydig cells, which are responsible for T production, would not only alter testicular function and feedback mechanisms, but would also have serious consequences for lean body mass. Indeed, there are indications in the literature that such damage induces changes in spermatogenesis (42, 43) and a decrease in T levels (44) in HIV patients, leading to cachexia.
In this study we show that MCP-1 and IP-10 are produced in abundance by both human and rat Leydig cells after exposure to the Sendai virus, whereas GRO synthesis was restricted to rat Leydig cells. MCP-1 levels were similar in rats and humans (135–280 ng MCP-1/µg DNA), suggesting that the chemokine system in Leydig cells is conserved in some mammals.
In conclusion, the Sendai virus induces the production of a number of chemokines, such as MCP-1, RANTES, GRO, and IP-10, in rat testicular macrophages and peritubular, Sertoli, and Leydig cells and in human Leydig cells, strongly suggesting that these somatic testicular cells, rather than germ cells, play a crucial role in leukocyte infiltration during viral infection of the testis.
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Acknowledgments |
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Footnotes |
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Abbreviations: EBV, Epstein-Barr virus; GRO, growth-related oncogene; HAU, hemagglutination units; HIV, human immunodeficiency virus; IFN, interferon-; IP-2, interferon--inducible protein-10; MCP-1, monocyte chemotactic protein-1; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted protein.
Received July 18, 2001.
Accepted for publication December 10, 2001.
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) or in the presence of 100 HAU/ml Sendai virus in allantoic fluid (+ virus; 
) for 0–72 h; or B) for 48 h in the presence of allantoic fluid (
) or in the presence of various concentrations (1–500 HAU/ml) of Sendai virus (
). MCP-1 production was quantified in culture media by ELISA. The data presented are the mean ± SEM for three wells, each assayed in duplicate, and are representative of three independent experiments.
) was quantified in culture media by specific ELISA. The data presented are the mean ± SEM for three wells, each assayed in duplicate, and are representative of three independent experiments.