Molecular Human Reproduction, Vol. 8, No. 6, 518-524,
June 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Expression of monocyte chemoattractant protein-1 and macrophage colony-stimulating factor in normal and inflamed rat testis
1 Monash Institute of Reproduction and Development, Monash University, 27–31 Wright Street, 2 Department of Nephrology and 3 Monash University Department of Medicine, Monash Medical Centre, Clayton 3168, Victoria, Australia
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Abstract |
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Macrophages are numerous in the testicular interstitial tissue under normal conditions and increase during inflammation. The mechanisms involved are poorly characterized. Expression of the macrophage-regulating cytokines monocyte chemoattractant protein (MCP)-1 and macrophage colony-stimulating factor (M-CSF) was examined in the adult rat testis before and after an i.p. injection of an inflammatory stimulus, lipopolysaccharide (LPS). In the normal testis, M-CSF was readily observed using Northern blot and Western blot analysis. In contrast, MCP-1 was not detectable by Northern blot in the normal testis, but was detected using RT–PCR amplification and a sensitive ELISA. After LPS treatment, testicular MCP-1 mRNA and protein expression increased dramatically (up to 400-fold). In-situ hybridization for MCP-1 revealed that production was confined to the interstitium of the inflamed testis, in Leydig cells, peritubular cells, perivascular cells and monocyte-like macrophages, but not in tissue-resident macrophages. Unlike MCP-1, M-CSF mRNA and protein expression in the testis increased only marginally, if at all, after LPS treatment. These results suggest that MCP-1 stimulates the increase in intratesticular macrophages that accompanies LPS-induced inflammation in vivo. Together with M-CSF, MCP-1 may also play a role in maintaining the resident macrophage population of the normal testis.
inflammation/M-CSF/MCP-1/monocytes/testis
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Introduction |
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Although the interstitial tissue of the rat testis is considered to be immunologically privileged, it is characterized by a large population of resident macrophages (Hutson, 1994
; Hedger, 1997). The testicular macrophages are deficient in inflammatory cytokine production in vitro, but have normal cell-killing and phagocytic functions (Wei et al., 1988; Kern et al., 1995; Hayes et al., 1996). These macrophages have also been shown to be involved in normal testicular function by regulating Leydig cell development and steroidogenesis (Hales et al., 1992; Bergh et al., 1993; Gaytan et al., 1994a,b). In turn, the accumulation of these cells is controlled principally by the Leydig cells, albeit through a mechanism that does not appear to directly involve androgens (Raburn et al., 1993; Wang et al., 1994; Duckett et al., 1997).
While maintenance of the testicular macrophage population, as in any other tissue, almost certainly involves the recruitment of circulating monocytes and their subsequent maturation into resident-type macrophages, the specific testicular signals responsible for regulating this process are very poorly defined. The only regulatory cytokine specific for macrophage development that definitely has been shown to play a role in controlling testicular macrophages is macrophage colony-stimulating factor (M-CSF). Previous studies have indicated that endogenous M-CSF regulates the number of resident testicular macrophages during testicular development (Cohen et al., 1996; Pollard et al., 1997). However, while there is some evidence for chemotactic activity of M-CSF in vitro (Wang et al., 1988), it is generally believed that the principal function of this pro-inflammatory cytokine is to regulate functional maturation and proliferation of macrophages rather than monocyte recruitment (Wiktor-Jedrzejczak et al., 1996).
Testicular macrophages can be identified immunohistochemically either by the expression of the resident macrophage marker ED2, or by the expression of ED1, a marker expressed on monocytes as well as macrophages (Dijkstra et al., 1985; Beelen et al., 1987; Wang et al., 1994). A small but significant number of testicular macrophages lack expression of the ED2 marker, suggesting that they may be circulating monocytes or newly-arrived macrophages that have not yet become resident (Wang et al., 1994).
We have found that a single i.p. injection of lipopolysaccharide (LPS) into adult male rats results in a marked influx of new monocytes (ED1+ED2-) into the testicular interstitium 12 h post-injection (Gerdprasert et al., 2002). In contrast, the number of resident (ED1-ED2+) testicular macrophages was not increased by the LPS treatment. Interestingly, the response to LPS in the testis was also characterized by a lack of granulocyte involvement, suggesting that a chemotactic substance specific to the monocyte population may be involved.
Among the known chemokine subfamilies, monocyte chemoattractant protein (MCP)-1 is the principal monocyte-selective chemotactic cytokine (Adams and Floyd, 1997; Lu et al., 1998). Significantly, this chemokine has been shown to be produced by several testicular interstitial cell types in vitro and is stimulated by LPS and inflammatory cytokines, suggesting that MCP-1 may be responsible for the recruitment of monocytes observed in vivo (Aubry et al., 2000). However, there is a possibility that the expression of MCP-1 by testicular cells in vitro, particularly the apparent `constitutive' expression in the absence of an inflammatory stimulus, could be an isolation or culture artefact. Moreover, the specific contribution of the testicular macrophage subsets to MCP-1 production was not examined in that study.
If MCP-1 is fundamentally involved in regulating monocyte recruitment in the rat testis, then one would expect to observe both endogenous expression of this chemokine in the normal testis in vivo, and a large up-regulation of MCP-1 prior to the increase in monocyte-macrophages in the LPS-induced inflammation model described above. Moreover, the emerging data that resident testicular macrophages are deficient in their pro-inflammatory response (Kern et al., 1995; Hayes et al., 1996) predicts that resident (ED2+) testicular macrophages should not participate to any significant degree in this production.
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Materials and methods |
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Experimental procedure
Experimental materials used in this study were generated as previously described (O'Bryan et al., 2000a
). Briefly, adult male Sprague-Dawley rats (110–120 days old, Monash University, Central Animal House, Clayton, Australia) received a single i.p. injection of pyrogen-free saline (control), or saline containing either 0.1 mg/kg body weight (low dose) or 5 mg/kg body weight (high dose) of LPS (from E. coli serotype 0127:B8; Sigma Chemical Company, St Louis, MO, USA). At various time points up to 72 h after injection the rats were anaesthetized with ether and the thorax and abdomen were exposed by a midline incision. A 1–2 ml sample of blood was collected by cardiac puncture. In some animals, one testis was removed for collection of testicular interstitial fluid (IF) as previously described (Hedger et al., 1990). Alternatively, one testis and a segment of liver were removed, snap-frozen and stored at –70°C for subsequent RNA or protein extraction. The remaining testis was fixed by whole-body perfusion with Bouin's fluid via the descending aorta. Excised tissues were post-fixed in Bouin's fluid for 3 h prior to being processed though standard paraffin techniques. All animal experiments were carried out in accordance with the National Health and Medical Research Council Guidelines on Ethics in Animal Experimentation, and were approved by Monash Medical Centre Animal Experimentation Ethics Committee.
RNA extraction and Northern blot analysis
Total RNA was isolated and purified from the testis and liver according to previously described methods (Chomczynski and Sacchi, 1987). Northern blotting was performed as previously described (O'Bryan et al., 2000b). Briefly, 
20 µg of testis and liver RNA from each sample was size-fractionated by electrophoresis on a 1.2% formaldehyde–agarose gel (BDH, Dorset, UK). RNA was transferred onto a Hybond N membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and hybridized sequentially with cDNA probes for rat MCP-1, kindly provided by Dr Teizo Yoshimura (Yoshimura et al., 1991), M-CSF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). cDNAs were labelled with 
32P-dATP using Strip-EZTM DNA probe synthesis kit (Ambion Inc., Austin, TX, USA). Blots were hybridized and washed using standard conditions. Hybridization was detected using a phosphoimager screen and read in a Fuji BAS1000 MacBAS Bio-Imaging Analyser (Fuji Photo Film Corporation, Tokyo, Japan) and quantified using the MacBAS V 2.5 software supplied by Fuji.
The intensity of MCP-1 and M-CSF expression was corrected for sample loading against the intensity of GAPDH expression for the same sample. The corrected data were then normalized against the data from saline-treated control rats, which were assigned an arbitrary value of 1.0. Experiments were repeated for a minimum of three animals per treatment group. Data were transformed to a normal distribution if necessary and analysed by one-way analysis of variance, followed by Dunnett's test for comparison of multiple means. All statistical analysis were performed using Sigmastat version 1.0 software (Jandel Corporation, San Rafael, CA, USA).
Semi-quantitative RT–PCR amplification
In order to determine if MCP-1 mRNA is expressed at very low levels in the normal testis, semi-quantitative RT–PCR was performed. mRNA was extracted from normal and LPS-treated testes (0–72 h post-injection) and cDNA was synthesized using the Omniscript RT kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. A 20 µl reaction contained 1 µmol/l Oligo-dT primer (Sigma-Genosys, Castle Hill, New South Wales, Australia), 10 units RNase inhibitor (Promega Corporation, Madison, WI, USA), 0.5 mmol/l dNTPs (Fisher Biotech, Perth, Australia), 2 µl 10xreverse transcriptase buffer (Qiagen) and 2 µg RNA. Reactions were incubated at 37°C for 60 min, 95°C for 5 min, then rapidly cooled on ice.
Preliminary experiments were performed to validate the quantitation of PCR products and to establish the conditions under which each amplification reaction was confined to the exponential phase of amplification, resulting in a yield of product proportional to the initial concentration of cDNA template (data not shown) (Wang et al., 1989).
PCR was performed using the following conditions: 30 s at 94°C, 30 s at 58°C and 1 min at 72°C for 29 cycles for GAPDH and 30 cycles for MCP-1. All PCR terminated with a final extension at 72°C for 10 min. Each 50 µl of master mix contained 1:50 dilution of cDNA, 10 pmol of GAPDH or 20 pmol of MCP-1 forward and reverse primers, 5 µl of 0.2 mmol/l dNTPs, 1.5 IU Taq polymerase and 5 µl of Taq polymerase buffer (Amersham). In order to minimize variation in the amplification process, master mixes were used in all reactions and each experiment was repeated three times with samples from different rats. All primers were obtained from Sigma-Genosys. The sequences of the forward and reverse primers for MCP-1 were 5'-CAGGTCTCTGTCACGCTTCT-3' and 5'-AGTATTCATGGAAGGGAATAG-3' respectively. The corresponding primers for GAPDH were 5'-ATCACTGCCACCCAGAAGACT-3' and 5'-CATGCCAGTGAGCTTCCCGTT-3'. Control reactions without cDNA were run in parallel for each reaction and were consistently negative in each case.
In-situ hybridization
The expression of MCP-1 mRNA within the rat testis was determined byin-situ hybridization with digoxigenin (DIG)-labelled antisense and sense MCP-1 probes using a previously described method (O'Bryan et al., 1998). Restriction enzymes for in-situ hybridization were obtained from New England Biolabs Inc. (Beverly, MA, USA). Briefly, labelled sense and antisense cRNA was synthesized by incubation of either HindIII (antisense) or XbaI (sense) linearized template—a plasmid containing MCP-1 CDNA—(1 µg) with DIG-labelled UTP (3.5 µmol/l) (Roche Diagnostics Corporation, Indianapolis, IN, USA) in the presence of T3 or T7 RNA polymerase (Promega), appropriate for the respective restriction enzyme employed, for 2 h at 37°C.
After synthesis, the amount of DIG-labelled RNA was determined by comparison with a DIG-labelled RNA standard (100 ng/ml; Roche). Testis sections were dewaxed and rehydrated though graded ethanol. Sections were treated with 0.2 mol/l HCl (BDH) for 20 min and partially digested with 5 µg/ml of proteinase K (Promega) for 30 min at 37°C. After incubation with 0.2% glycine (Sigma) for 10 min at 4°C, sections were acetylated in 0.25% acetic anhydride (Sigma) in the present of 0.1 mol/l triethanolamine (BDH) for 5 min and prehybridized in prehybridization buffer [50% deionized formamide, 3xsaline sodium citrate (SSC), 1xDenhardt's solution and 0.2 mol/l phosphate buffer] for a minimum of 2 h at 50°C.
Hybridization was performed overnight at 50°C in prehybridization buffer containing 10% dextran sulphate (Pharmacia Biotech), 1 mg/ml yeast total RNA (Promega), 1 mg/ml herring sperm DNA (Roche) and 200 ng/ml of MCP-1 DIG-labelled probe. Unbound probe was removed by 20 µg/ml RNase A (Roche) in 2xSSC at 37°C for 30 min followed by sequential washes in SSC solutions to a maximum stringency of 0.1xSSC at 50°C. Hybridized probe was localized on tissue sections using an anti-DIG alkaline phosphatase-conjugated antibody (dilution 1:1000; Roche). Non-specific antibody binding was reduced by pre-incubating sections in DIG-blocking solution for 30 min at room temperature. Specifically bound riboprobes were visualized by an enzyme-catalysed colour reaction using nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate substrate (NBT/BCIP one step; Pierce Chemical Company, Rockford, IL, USA). Sections were mounted under glass coverslips using GVA histomount (Zymed, San Franscisco, CA, USA). In order to differentiate testicular cells, slides were counterstained with Mayer's haematoxylin before mounting.
Immunohistochemistry
In order to identify testicular macrophages, and to differentiate testicular macrophage subsets after MCP-1 in-situ hybridization, testis sections were washed in 0.01 mol/l phosphate-buffered saline (PBS), pH 7.4, and subsequently processed for immunohistochemistry with ED1 and/or ED2 using a modification of a method described previously (Schlatt et al., 1999). Briefly, sections were incubated in 0.25 mol/l glycine–HCl buffer (pH 3.5) in a microwave oven at 2.25 W/ml/min for 3 min and left to cool. The slides were subsequently washed in PBS and pre-incubated with 10% normal sheep serum for 30 min, and then incubated overnight at 4°C with either ED1 or ED2 antibody (supernatants from cultures of hybridomas supplied by the ECACC, Porton Down, UK) alone or together, or mouse serum as a negative control diluted in PBS containing 1% normal sheep serum and 0.01% bovine serum albumin (BSA; Sigma). After washing, the sections were incubated at room temperature for 1 h with a biotin-conjugated sheep-anti-mouse IgG serum (diluted 1:250; Silenus, Amrad Biotech, Boronia, Victoria, Australia). The sections were washed and further incubated for 1 h with streptavidin-conjugated alkaline phosphatase (diluted 1:250; Silenus). Excess antibodies were removed by washing in PBS. Alkaline phosphatase activity was visualized using Fast Red (Sigma). Sections were mounted using Ultramount (Dako Corporation, Carpinteria, CA, USA).
Protein extraction
Testis samples were homogenized in 5 mmol/l PBS, 1% NP-40, 0.5% Tween 20 and 0.1% sodium dodecyl sulphate (SDS), containing a broad spectrum proteinase inhibitor cocktail (Boehringer, Mannheim, Germany). The protein contents of the resulting testis extracts were determined using the BioRad DC protein assay (BioRad, Hercules, CA, USA).
Rat MCP-1 ELISA
Concentrations of MCP-1 were measured in testis extracts and IF using a rat MCP-1 OptiEIA ELISA kit (BD PharMingen, Los Angeles, CA, USA). The assay standard was a recombinant rat MCP-1 (Yoshimura et al., 1991) and the capture and detection antibody was a mouse monoclonal antibody raised against rat MCP-1 (Sakanashi et al., 1994). The assay had a sensitivity of <30 pg/ml. All samples were assayed at a minimum of three serial dilutions and assessed against the standard using routine parallel-line bioassay statistics.
M-CSF Western blot procedure
Expression of M-CSF in the rat testis was determined by Western blot analysis using a previously described protocol (Isbel et al., 2001). Briefly, testicular IF and serum samples were mixed with sample buffer (60 mmol/l Tris–HCl, pH 6.8, 2% SDS, 20% v/v glycerol, 0.01% Bromophenol Blue, 0.05% ß-mercaptoethanol), boiled for 5 min and separated electrophoretically on a 12.5% SDS polyacrylamide gel. Proteins were transferred onto Hybond ECL nitrocellulose membrane (Amersham) with a BioRad Transblot overnight. The membrane was blocked for 2 h in PBS containing 5% skimmed milk powder, 2% BSA and 0.05% Tween 20, then incubated for 2 h with 25 µg/ml guinea pig anti-rat M-CSF antibody (Isbel et al., 2001). After washing with PBS containing 0.05% Tween 20, the membrane was incubated for 1 h with a peroxidase-conjugated rabbit anti-guinea pig IgG (Dako), diluted 1:20 000 in blocking buffer. The membrane was washed and developed using SuperSignal Chemiluminescent substrate (Pierce). The signal was captured on Biomax film (Kodak, Melbourne, Australia).
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Macrophages in normal and LPS-treated testis
Treatment of adult male rats with either a low dose (0.1 mg/kg) or high dose (5 mg/kg) of LPS caused an increase in monocytes and macrophages in the testicular interstitial tissue, peaking at 12 h after the LPS-injection (Figure 1
). Monocytes and macrophages were never observed within the limiting basement membrane of the seminiferous tubules, even in the high dose LPS-treated animals.
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Expression of MCP-1 and M-CSF mRNA in normal and LPS-treated testis
As measured by Northern blotting, MCP-1 mRNA was undetectable in control (0 h) testis and liver samples (Figure 2A
). The level of MCP-1 mRNA (a band of 1.0 kb) increased dramatically at 3–6 h after either low or high dose of LPS in both the testis and liver. The real fold increase over control could not be quantified, because there was no measurable expression in the control samples, but the peak expression of MCP-1 mRNA in the testis at both LPS doses was at least as great, and possibly even greater than that observed in the liver. Peak expression levels in each tissue were not different between the high and low dose groups, but expression in the testis was more prolonged in the high dose group. Levels had returned to close to normal by 12 h after injection in both tissues, although there was a detectable signal in some samples up to 72 h later, and particularly at 18 h after LPS injection.
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In contrast to MCP-1, M-CSF mRNA (a band of
4 kb) was clearly present in both normal control testis and liver (Figure 2B
). The expression of M-CSF in the testis increased only slightly (2-fold) over controls at 3 h after injection of LPS, whereas liver expression increased more substantially (3- to 5-fold over 3–6 h).
Expression of MCP-1 mRNA by semi-quantitative RT–PCR
Using the more sensitive, semi-quantitative technique of RT–PCR amplification, MCP-1 mRNA was detected at relatively low levels in control testes compared with samples from LPS-treated rats (Figure 3). This experiment was repeated with a total of three different animals at each time point, with similar results.
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Cellular localization of MCP-1 by in-situ hybridization
In order to localize MCP-1 mRNA in the testis, in-situ hybridization using a MCP-1 cRNA antisense probe was performed on testis sections collected from control and LPS-treated rats. The sense MCP-1 cRNA control probe did not show any staining in sections of normal or LPS-treated testis at any time point investigated (Figure 4A
), confirming the specificity of this technique. Using the antisense MCP-1 probe, mRNA was not detectable in normal, saline-injected testes (Figure 4B), nor in testes collected at 24 h after LPS treatment (results not shown). However, MCP-1 mRNA was detectable within the interstitium of the testis after 3 and 6 h in both high and low dose LPS-treated rats (Figure 4C). No signal was seen within germ cells and Sertoli cells at any time point. Staining was confined to the interstitial tissues, localized to peritubular cells, perivascular (endothelial and smooth muscle) cells, fibroblast-like cells and the Leydig cells (Figure 4D). Using immunohistochemistry in conjunction with MCP-1 in-situ hybridization, MCP-1 was frequently observed in ED1+ macrophages (Figure 4E), but rarely in ED2+ tissue-resident macrophages (Figure 4F) in the LPS-treated testes.
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Measurement of MCP-1 and M-CSF protein in normal and LPS-treated testis
The concentration of MCP-1 in IF and testicular extracts from normal rats measured by specific ELISA were 38.3 ± 8.3 ng/ml (mean ± SEM, n = 5 animals) and 0.075 ± 0.014 ng/mg protein (4.6 ± 0.9 ng/testis) respectively. At 6 h after treatment with LPS, MCP-1 protein levels in testis extracts increased by an average of 20-fold (low dose group) and 400-fold (high dose group) (Figure 5
).
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M-CSF protein levels as measured by Western blot were similar in serum and testicular IF from normal rats, and LPS did not appear to cause any substantial up-regulation of protein expression in the testis at either treatment dose (Figure 6
).
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Discussion |
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This study establishes that MCP-1, as detected by mRNA and protein expression, is present in the normal rat testis at low, but apparently physiologically relevant, levels. These data also confirm that the baseline expression of MCP-1 by testicular cells in culture as reported by Aubry et al. was due to constitutive expression, not simply to activation by the isolation procedures (Aubry et al., 2000
). The relative amount of MCP-1 protein in whole testis extracts and testicular IF from normal rats indicated a preferential concentration of MCP-1 in the fluid compartment of the interstitial tissue of 38 ng/ml (4 nmol/l). Consequently, the levels of MCP-1 in the testis of normal rats appear to be very close to the ED50 required for significant monocyte chemotaxis by mouse MCP-1 in vitro (Ernst et al., 1994; Luo et al., 1994). This observation supports at least some role for this cytokine in maintaining the normal macrophage population.
Following an i.p. injection of either a low dose or high dose of LPS, expression of MCP-1 mRNA in the testis was up-regulated to levels comparable with those observed in the liver. The up-regulation of MCP-1 mRNA occurred largely within the first 6 h after treatment, and was accompanied by a corresponding dose-dependent increase of testicular MCP-1 protein levels of 20- and 400-fold at 6 h in the low and high dose treatment groups respectively. Since this increase in MCP-1 expression immediately preceded a large influx of monocytes into the testicular interstitial tissue, these data clearly suggest that MCP-1 contributes to monocyte recruitment in the inflamed rat testis. Although there was also a slight apparent increase in MCP-1 mRNA at 18 h after LPS treatment, the significance of this is less certain since it was not accompanied by a detectable increase in monocytes (Gerdprasert et al., 2002).
Using in-situ hybridization to identify cellular sites of production, expression of MCP-1 in the inflamed testis was confined to the interstitial space, in peritubular cells, perivascular cells, fibroblasts, macrophages and Leydig cells. Germ cells and Sertoli cells showed no detectable expression following either dose of LPS. The expression in fibroblasts, macrophages, peritubular cells and perivascular cells is consistent with the well characterized expression of MCP-1 by fibroblasts, macrophages and smooth muscle cells in many other tissues (Wiktor-Jedrzejczak et al., 1996). The testis-specific pattern of expression by the Leydig cells, but not by cells of the seminiferous tubules, confirms recent data obtained from isolated testicular cell subsets in vitro (Aubry et al., 2000). The fact that MCP-1 is not expressed by the cells of the seminiferous tubules is consistent with the fact that monocyte infiltration is confined to the interstitial compartment of the testis during LPS-induced inflammation (O'Bryan et al., 2000a).
Using a double-labelling procedure which allowed the simultaneous detection of MCP-1 mRNA and antibodies for testicular macrophage subset markers, MCP-1 expression was found in some ED1+ monocytes or macrophages, but not in ED2+ resident macrophages. These data are further support for the emerging concept that the resident testicular macrophages lack the capacity for production of pro-inflammatory cytokines and mediators (Kern et al., 1995; Hayes et al., 1996; Gerdprasert et al., 2002). Curiously, this lack of production of MCP-1 co-exists with expression of the cytokine by the Leydig cells, which are of fibroblastic rather than haematopoietic derivation, as well as by peritubular cells, smooth muscle cells and endothelial cells. Recently, we have shown that expression of another pro-inflammatory cytokine, interleukin (IL)-1ß, is also up-regulated within the testis during inflammation (Gow et al., 2001), although the cellular site of production was not investigated in that study. While it is uncertain how the local environment of the testis brings about down-regulation of inflammatory capacity of the testicular macrophages, these data suggest that regulation may be specific to the cells of the monocyte/macrophage lineage, and that other testicular cells retain their capacity to produce inflammatory mediators, including cytokines.
M-CSF has numerous affects on the development of the resident macrophage population in all tissues (Stanley et al., 1997). It has been shown that male mice lacking M-CSF have greatly reduced testicular macrophage numbers and impaired fertility (Cohen et al., 1996; Pollard et al., 1997). Both parameters can be restored by treatment with exogenous M-CSF, indicating that this cytokine plays an important role in development of the testicular macrophage population and in spermatogenesis. In the present study, M-CSF displayed a much higher constitutive level of mRNA expression in the rat testis than MCP-1, which was only detectable by RT–PCR in the normal testis. Moreover, M-CSF mRNA was expressed in the normal rat testis at levels comparable with those in the liver, another tissue containing numerous resident macrophages. This supports data from the mouse that M-CSF is expressed in the normal testis (Cohen et al., 1996), and suggests an ongoing role for M-CSF in maintaining the testicular macrophage phenotype in the adult.
In contrast to MCP-1, however, M-CSF mRNA expression was only very slightly increased by LPS treatment. There was no available assay for rat M-CSF at the time of this study, hence protein levels were measured using Western blot analysis. The data showed that the levels of M-CSF protein in the testicular IF were comparable with the levels seen in normal circulation, and confirmed the Northern blot data which indicated that there was little or no increase in M-CSF in the testis after LPS treatment. These data suggest that M-CSF, certainly in contrast to MCP-1, plays no significant role in macrophage recruitment during testicular inflammation caused by LPS.
In conclusion, this study clearly suggests a key role for MCP-1 in recruiting macrophages to the testis during inflammation, as well as a minor role in stimulating macrophage accumulation and development in the testis under normal conditions. Nonetheless, the relative levels of mRNA expression suggest that M-CSF is the more important cytokine with regard to regulation in the normal testis. This relative importance is further supported by the fact that, while M-CSF-deficient male mice have reduced fertility due to a reduction in testicular resident macrophages and resulting Leydig cell dysfunction (Cohen et al., 1996), there have been no reports of obvious male fertility problems in mice in which the MCP-1/receptor axis has been inactivated (Boring et al., 1997; Kurihara et al., 1997). Finally, MCP-1 also represents one more pro-inflammatory marker, together with IL-1ß, tumour necrosis factor- and inducible nitric oxide synthase, for which the resident testicular macrophage population displays a poor capacity for production (Kern et al., 1995; Hayes et al., 1996; Gerdprasert et al., 2002). Consequently, these cytokines represent important investigative foci for future studies aimed at understanding the unique regulation and functions of the testicular macrophage population.
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The authors thank Ms Rita Foti for expert technical assistance. These studies were supported by grants from the National Health and Medical Research Council of Australia (grant no. 973218 and 143781), and a Royal Thai Government Scholarship (to Dr Gerdprasert).
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4 To whom correspondence should be addressed. E-mail: mark.hedger{at}med.monash.edu.au
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References |
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Adams, D.H. and Lloyd, A.R. (1997) Chemokines: leucocyte recruitment and activation cytokines. Lancet, 349, 490–495.[ISI][Medline]
Aubry, F., Habasque, C., Satie, A.P., Jegou, B. and Samson, M. (2000) Expression and regulation of the CC-chemokine monocyte chemoattractant protein-1 in rat testicular cells in primary culture. Biol. Reprod., 62, 1427–1435.
Bergh, A., Damber, J.E. and van Rooijen, N. (1993) Liposome-mediated macrophage depletion: an experimental approach to study the role of testicular macrophages in the rat. J. Endocrinol., 136, 407–413.[Abstract]
Boring, L., Gosling, J., Chensue, S.W., Kunkel, S.L., Farese, R.V. Jr, Broxmeyer, H.E. and Charo, I.F. (1997) Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice. J. Clin. Invest., 100, 2552–2561.[ISI][Medline]
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem., 162, 156–159.[ISI][Medline]
Cohen, P.E., Chisholm, O., Arceci, R.J., Stanley, E.R. and Pollard, J.W. (1996) Absence of colony-stimulating factor-1 in osteopetrotic (csfm°p/csfm°p) mice results in male fertility defects. Biol. Reprod., 55, 310–317.[Abstract]
Damaoiseaux, J.G., Dopp, E.A., Neefjes, J.J. and Dijkstra, C. (1989) Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new macrophage antibodies against a heterodimer of 160 and 95 kd (CD11/CD18). J. Leukoc. Biol., 46, 556–564.[Abstract]
Dijkstra, C.D., Dopp, E.A., Joling, P. and Kraal, G. (1985) The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3. Immunology, 54, 589–599.[ISI][Medline]
Duckett, R.J., Wreford, N.G., Meachem, S.J., McLachlan, R.I. and Hedger, M.P. (1997) Effect of chorionic gonadotropin and flutamide on Leydig cell and macrophage populations in the testosterone–estradiol-implanted adult rat. J. Androl., 18, 656–662.
Ernst, C.A., Zhang, Y.J., Hancock, P.R., Rutledge, B.J., Corless, C.L. and Rollins, B.J. (1994) Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains. J. Immunol., 152, 3541–3549.[Abstract]
Gaytan, F., Bellido, C., Aguilar, E. and van Rooijen, N. (1994a) Requirement for testicular macrophages in Leydig cell proliferation and differentiation during prepubertal development in rats. J. Reprod. Fertil., 102, 393–399.[Abstract]
Gaytan, F., Bellido, C., Morales, C., Reymundo, C., Aguilar, E. and Van Rooijen, N. (1994b) Effects of macrophage depletion at different times after treatment with ethylene dimethane sulfonate (EDS) on the regeneration of Leydig cells in the adult rat. J. Androl., 15, 558–564.
Gerdprasert, O., O'Bryan, M.K., Muir, J.A., Caldwell, A.M., Schlatt, S., de Kretser, D.M. and Hedger, M.P. (2002) The response of testicular leukocytes to lipopolysaccharide-induced inflammation: evidence for functional heterogeneity of the testicular macrophage population. Cell Tissue Res., in press.
Gow, R.M., O'Bryan, M.K., Canny, B.J., Ooi, G.T. and Hedger, M.P. (2001) Differential effects of dexamethasone treatment on lipopolysaccharide-induced testicular inflammation and reproductive hormone inhibition in adult rats. J. Endocrinol., 168, 193–201.[Abstract]
Hales, D.B., Xiong, Y. and Tur-Kaspa, I. (1992) The role of cytokines in the regulation of Leydig cell P450c17 gene expression. J. Steroid Biochem. Mol. Biol., 131, 2165–2172.
Hayes, R., Chalmers, S.A., Nikolic-Paterson, D.J., Atkins, R.C. and Hedger, M.P. (1996) Secretion of bioactive interleukin 1 by rat testicular macrophages in vitro. J. Androl., 17, 41–49.
Hedger, M.P. (1997) Testicular leukocytes: what are they doing? Rev. Reprod., 2, 38–47.[Abstract]
Hedger, M.P., Robertson, D.M., de Kretser, D.M. and Risbridger, G.P. (1990) The quantification of steroidogenesis-stimulating activity in testicular interstitial fluid by an in vitro bioassay employing adult rat Leydig cells. Endocrinology, 127, 1967–1977.[Abstract]
Hutson, J.C. (1994) Testicular macrophages. Int. Rev. Cytol., 149, 99–143.[ISI][Medline]
Isbel, N.M., Hill, P.A., Foti, R., Mu, W., Hurst, L.A., Stambe, C., Lan, H.Y., Atkins, R.C. and Nikolic-Paterson, D.J. (2001) Tubules are the major site of M-CSF production in experimental kidney disease: Correlation with local macrophage proliferation. Kidney Int., 60, 614–625.[ISI][Medline]
Kern, S., Robertson, S.A., Mau, V.J. and Maddocks, S. (1995) Cytokine secretion by macrophages in the rat testis. Biol. Reprod., 53, 1407–1416.[Abstract]
Kurihara, T., Warr, G., Loy, J. and Bravo, R. (1997) Defects in macrophage recruitment and host defence in mice lacking the CCR2 chemokine receptor. J. Exp. Med., 186, 1757–1762.
Lu, B., Rutledge, B.J., Gu, L., Fiorillo, J., Lukacs, N.W., Kunkel, S.L., North, R., Gerard, C. and Rollins, B.J. (1998) Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J. Exp. Med., 187, 601–608.
Luo, Y., Laning, J., Hayashi, M., Hancock, P.R., Rollins, B. and Dorf, M.E. (1994) Serologic analysis of the mouse ß chemokine JE/monocyte chemoattractant protein-1. J. Immunol., 153, 3708–3716.[Abstract]
O'Bryan, M.K., Loveland, K.L., Herszfeld, D., McFarlane, J.R., Hearn, M.T. and de Kretser, D.M. (1998) Identification of a rat testis-specific gene encoding a potential rat outer dense fibre protein. Mol. Reprod. Dev., 50, 313–322.[ISI][Medline]
O'Bryan, M.K., Schlatt, S., Phillips, D.J., de Kretser, D.M. and Hedger, M.P. (2000a) Bacterial lipopolysaccharide-induced inflammation compromises testicular function at multiple levels in vivo. Endocrinology, 141, 238–246.
O'Bryan, M.K., Sebire, K.L., Gerdprasert, O., Hedger, M.P., Hearn, M.T. and de Kretser, D.M. (2000b) Cloning and regulation of the rat activin ßE subunit. J. Mol. Endocrinol., 24, 409–418.[Abstract]
Pollard, J.W., Dominguez, M.G., Mocci, S., Cohen, P.E. and Stanley, E.R. (1997) Effect of the colony-stimulating factor-1 null mutation, osteopetrotic (csfm°p), on the distribution of macrophages in the male mouse reproductive tract. Biol. Reprod., 56, 1290–1300.[Abstract]
Raburn, D.J., Coquelin, A., Reinhart, A.J. and Hutson, J.C. (1993) Regulation of the macrophage population in postnatal rat testis. J. Reprod. Immunol., 24, 139–151.[ISI][Medline]
Sakanashi, Y., Takeya, M., Yoshimura, T., Feng, L., Morioka, T. and Takahashi, K. (1994) Kinetics of macrophage subpopulations and expression of monocyte chemoattractant protein-1 (MCP-1) in bleomycin-induced lung injury of rats studied by a novel monoclonal antibody against rat MCP-1. J. Leuk. Biol., 56, 741–750.[Abstract]
Schlatt, S., de Kretser, D.M. and Hedger, M.P. (1999) Mitosis of resident macrophages in the adult rat testis. J. Reprod. Fertil., 116, 223–228.[Abstract]
Stanley, E.R., Berg, K.L., Einstein, D.B., Lee, P.S., Pixley, F.J., Wang, Y. and Yeung, Y.G. (1997) Biology and action of colony-stimulating factor-1. Mol. Reprod. Dev., 46, 4–10.[ISI][Medline]
Wang, J.M., Griffin, J.D., Rambaldi, A., Chen, Z.G. and Mantovani, A. (1988) Induction of monocyte migration by recombinant macrophage colony-stimulating factor. J. Immunol., 141, 575–579[Abstract]
Wang, A.M., Doyle, M.V. and Mark, D.F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl Acad. Sci. USA, 86, 9717–9721.
Wang, J., Wreford, N.G., Lan, H.Y., Atkins, R. and Hedger, M.P. (1994) Leukocyte populations of the adult rat testis following removal of the Leydig cells by treatment with ethane dimethane sulfonate and subcutaneous testosterone implants. Biol. Reprod., 51, 551–561.[Abstract]
Wei, R.Q., Yee, J.B., Straus, D.C. and Hutson, J.C. (1988) Bactericidal activity of testicular macrophages. Biol. Reprod., 38, 830–835.[Abstract]
Wiktor-Jedrzejczak, W. and Gordon, S. (1996) Cytokine regulation of the macrophage (M
) system studied using the colony stimulating factor-1-deficient op/op mouse. Physiol. Rev., 76, 927–947.
Yoshimura, T., Takeya, M. and Takahashi, K. (1991) Molecular cloning of rat monocyte chemoattractant protein-1 (MCP-1) and its expression in rat spleen cells and tumor cell lines. Biochem. Biophys. Res. Comm., 174, 504–509.[ISI][Medline]
Submitted on May 15, 2001; resubmitted on December 28, 2001; accepted on March 6, 2002.
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