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Endocrinology Vol. 139, No. 8 3637-3645
Copyright © 1998 by The Endocrine Society

ARTICLES

Expression and Regulation of Interferon-{gamma}-Inducible Protein 10 Gene in Rat Leydig Cells1

Jianxin Hu, Shaojin You, Wei Li, Deli Wang, Madan L. Nagpal, Yide Mi, Peng Liang and Tu Lin

Research and Medical Service, WJB Dorn Veterans Medical Center, and the Department of Medicine, University of South Carolina School of Medicine (J.H., S.Y., W.L., D.W., M.L.N., Y.M., T.L.), Columbia, South Carolina 29208; and the Vanderbilt Cancer Center, Department of Cell Biology, Vanderbilt University School of Medicine (P.L.), Nashville, Tennessee 37232

Address all correspondence and requests for reprints to: Tu Lin, M.D., Department of Medicine, University of South Carolina School of Medicine, Medical Library Building, Suite 316, Columbia, South Carolina 29208.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we report the cloning of a gene that is differentially expressed in normal adult rat Leydig cells and whose expression is inhibited by hCG but is induced by interferon-{gamma} (IFN{gamma}). DNA sequence analysis identified this gene as rat IFN{gamma}-inducible protein 10 (IP-10), a member of the -C-X-C- chemokine superfamily of proinflammatory cytokines. High levels of IP-10 messenger RNA (mRNA) were constitutively expressed in freshly isolated and primary cultured Leydig cells. hCG inhibited this expression in a dose-dependent manner. The addition of 1 ng/ml hCG inhibited IP-10 mRNA levels more than 80%. Conversely, IP-10 mRNA levels were markedly increased in response to murine interleukin-1{alpha}, murine tumor necrosis factor-{alpha}, and murine IFN{gamma} by 3.3-, 10-, and 26-fold, respectively. Concomitant addition of murine interleukin-1{alpha}, murine tumor necrosis factor-{alpha}, and murine IFN{gamma} synergistically increased IP-10 mRNA levels by 58-fold. Furthermore, in addition to one previously described rat IP-10 mRNA transcript (1.5 kb), another larger transcript (2.7 kb) was identified by Northern blot in rat Leydig cells. After screening a rat testis complementary DNA library, we obtained a partial structural gene and an intron sequence, which possibly originated from the larger transcript of rat IP-10 mRNA. Histochemical and immunocytochemical staining revealed that purified cells were positive for 3ß-hydroxysteroid dehydrogenase and IP-10, confirming that IP-10 is indeed present in Leydig cells. IP-10 antisense oligonucleotides enhanced basal and hCG-induced testosterone formation. This suggests that endogenous IP-10 has an inhibitory effect on Leydig cell steroidogenesis. In conclusion, IP-10 is expressed in rat Leydig cells and may have paracrine and autocrine effects on testicular function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAJOR physiological role of Leydig cells is to produce androgen required for spermatogenesis. In recent years, it has become clear that Leydig cells also produce a variety of peptides, growth factors, and cytokines (e.g. activin, inhibin, angiotensin II, CRH, POMC, GH-releasing hormone, and insulin-like growth factor I), which may have paracrine or autocrine effects on testicular function (for review, see Refs. 1, 2). Among the various cytokines, interleukin-1 (IL-1) has been the most extensively studied in Leydig cells. We reported previously that both IL-1{alpha} and IL-1ß messenger RNAs (mRNAs) are expressed in IL-1ß-stimulated rat Leydig cells, with IL-1ß being the predominant species (3, 4). Furthermore, IL-1ß mRNA can be induced by a single injection of hCG in vivo (4). IL-1 inhibits Leydig cell steroidogenesis (5, 6, 7, 8, 9), but may act as a growth factor for spermatogonia and regulate spermatogenesis (10, 11). Furthermore, IL-1 has a mitogenic effect on immature Leydig cells (12). IL-6 mRNA is also expressed in purified Leydig cells, and its secretion is enhanced by hCG and IL-1ß (13). IL-6 affected the secretion of transferrin from Sertoli cells in both an acute and a chronic fashion (14). Here we report the identification of a gene, constitutively expressed in rat Leydig cells, that is inhibited by hCG and induced by interferon-{gamma} (IFN{gamma}). A GenBank search identified this gene as a chemokine, IFN-inducible protein 10 (IP-10) (15, 16). Based on sequence, the chemokine family could be divided into two groups: {alpha}-chemokines, also known as -C-X-C- chemokines, contain a single amino acid between the first and second cysteine residues; the ß, or -C-C-, chemokines have adjacent cysteine residues (17, 18). Chemokines are active as chemotactic factors and growth regulators, and exert their effects through seven transmembrane domain G protein-coupled receptors (18). IP-10 belongs to the -C-X-C- (or {alpha}) chemokine superfamily, and it is 31% identical to platelet factor-4 and 26% identical to IL-8, two other members of the -C-X-C- chemokine family (17, 18).mob-1, by sequence homology, is the rat homolog of IP-10 (19, 20). IP-10/mob-1 is a downstream target of the Ras signaling pathway that is involved in cell proliferation (19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
RNAmap kit was from GenHunter Co. (Brookline, MA). The TA cloning kit was obtained from Invitrogen Co. (San Diego, CA). AmpliTaq DNA polymerase was purchased from Perkin-Elmer (Norwalk, CT). The Sequenase version 2.0 DNA Sequencing Kit was obtained from U.S. Biochemical Corp. (Cleveland, OH). The rat testis 5'-stretch plus complementary DNA (cDNA) library was from Clontech Laboratories (Palo Alto, CA). [{alpha}-35S]Deoxy (d)-ATP (1000 Ci/mmol) and [{alpha}-32P]dCTP (3000 Ci/mmol) were purchased from ICN Biochemical (Costa Mesa, CA). Recombinant murine IL-1{alpha} (mIL-1{alpha}), murine tumor necrosis factor-{alpha} (mTNF-{alpha}), and murine IFN{gamma} (mIFN{gamma}) were purchased from Genzyme Corp. (Cambridge, MA). Recombinant human IP-10 was obtained from Pepro Tech (Rocky Hill, NJ). The highly purified hCG (13,000 U/mg) was provided by Dr. Patricia Morris (The Population Council, Rockefeller University, New York, NY). Rat IP-10/mob-1 antibody was prepared as reported previously (19).

Isolation and culture of rat Leydig cells
Normal male Sprague-Dawley rats (55–65 days old; Charles River, Raleigh, NC) were killed after asphyxiation in a CO2-precharged chamber, and testes were removed. All procedures were carried out aseptically. The method of Klinefelter et al. (21), with minor modifications, was then used to isolate highly purified Leydig cells (22). Using 3ß-hydroxy-steroid dehydrogenase (3ßHSD) staining, more than 97% of the purified cells stained positive. As 20% of interstitial cells are macrophages, it is important to document that Leydig cell preparations were not contaminated with macrophages, which are a rich source of cytokines. Using mouse antirat macrophage monoclonal antibody (ED2), we found previously that less than 1% of a purified Leydig cell preparation stained positive for macrophages (4). Purified Leydig cells were cultured in DMEM-Ham’s F-12 with 0.5% BSA, 15 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin for 24 h. After medium change, cells were treated with or without hCG (10 ng/ml) for 6 h. Total cellular RNA was then extracted.

Isolation of total RNA
Total cellular RNA was extracted according to the method of Chomczynski and Sacchi (23) with minor modifications, as described previously (24). The purity and the yield of isolated RNA were determined by monitoring absorbance at 260 and 280 nm. The integrity of the RNA was confirmed by performing denaturing agarose gel electrophoresis on the isolated RNA samples.

Differential display PCR
Differential display RT-PCR was carried out using the RNA-map kit (GenHunter, Brookline, MA) as instructed by the manufacturer. Briefly, four RT reactions were conducted, each containing a different T12 MN (10 µM) 3'-primer, 2 µl total RNA (0.1 µg/µl), 4 µl 5 x RT buffer, 1.6 µl dNTP (250 µM), and 9.4 µl ribonuclease (RNase)-free distilled H2O. Samples were cycled (The MiniCycler, model PTC-150, MJ Research, Watertown, MA) at 65 C for 5 min and cooled to 37 C for 10 min. One hundred units of Moloney murine leukemia virus RT were added, and samples were incubated for an additional 60 min at 37 C, heated to 95 C for 5 min, cooled to 4 C, and stored at -20 C. Four separate PCR reactions were conducted with 9.2 µl distilled H2O, 2 µl 10 x PCR buffer, 1.6 µl dNTP (25 µM), 2 µl activating protein-2 (AP-2) arbitrary primer (5'-GACCGCTTGT-3'; 2 µM), 2 µl T12 MN (10 µM, corresponding to that used during RT), 2 µl RT mix from above, 1 µl [{alpha}-35S]dATP (1000 Ci/mmol), and 0.2 µl AmpliTaq (Perkin-Elmer) for a final reaction volume of 20 µl. The reaction was overlaid with 25 µl mineral oil and reaction cycled as follows: denatured at 94 C for 30 sec, annealed at 40 C for 2 min, and extended at 72 C for 30 sec for 40 cycles, followed by a 5-min polish at 72 C. Two microliters of loading dye were added to 3.5 µl of the sample and heated for 2 min at 80 C before loading onto a 6% denaturing acrylamide sequencing gel. Electrophoresis was performed for 2.5 h at 6 watts, and the gel was transferred to Whatman paper (Whatman, Clifton, NJ) and dried under vacuum at 80 C. The dried gel was then exposed to Fuji RX x-ray film (Fuji, Tokyo, Japan) for 48–72 h.

Amplified transcripts that consistently scored as differentially expressed in hCG-positive or -negative lanes from multiple experiments were precisely excised from the filter blot and incubated in distilled H2O for 10 min at room temperature and in a boiling water bath for 15 min. DNA was ethanol precipitated, with glycogen added as a carrier. DNA was reamplified using the appropriate primer pairs under identical PCR conditions containing 250 µM dNTP stock and minus isotope. An aliquot of the PCR reaction was directly ligated into the pCR-II TA vector (Invitrogen, San Diego, CA) following the specifications supplied by the manufacturer. The clones were then sequenced by the chain termination reactions on double stranded plasmid DNA with the Sequenase kit (U.S. Biochemical). The clone containing an insert corresponding to rat IP-10 gene downstream sequence was named pA21.

RT-PCR amplification of IP-10 probe
Total RNA (0.2 µg) from normal primary cultured Leydig cells was reverse transcribed using T12 MA primer by the method described for differential display PCR. Two microliters of RT mix were then PCR amplified using two primers. The upstream primer (5'-ATGAACCCAAGTGCTGCCGTCATT-3') was based on the structural gene sequence of murine IP-10, and the downstream primer (5'-CTACCCATTGATACATAC-3') was based on the sequence of the insert of pA21. The PCR product was cloned into pCR-II TA vector (recombinant was termed as pRLG17) and sequenced by the method described above.

Northern blot analysis
Twenty micrograms of total RNA from each sample were denatured with 6% formaldehyde and 50% formamide, run on a 1% agarose gel containing 2.2 M formaldehyde, and then transferred onto Nytran membrane (0.45 µm; Schleicher and Schuell, Keene, NH) by capillary elution. The nucleic acids were immobilized on the Nytran membrane by UV cross-linking (Stratalinker UV Crosslinker, Stratagene, La Jolla, CA). The membrane was prehybridized at 65 C for at least 4 h in a mixture of 7% SDS, 0.25 M NaH2PO4, 0.1% BSA, and 1 mM EDTA, pH 8.0, in a hybridization incubator (model 310, Robbins Scientific Corp., Sunnyvale, CA). Hybridization was carried out in the same solution at 65 C overnight with 3 ml hybridization buffer and IP-10 probe labeled with [{alpha}-32P]dCTP (3000 Ci/mmol) using a Random Primers DNA Labeling System (Life Technologies, Gaithersburg, MD). The membrane was washed three times with 200 ml wash solution (0.1 x SSC and 0.1% SDS) at 65 C and then exposed to Fuji RX x-ray film with an intensifying screen at -70 C. The autoradiograms were quantified by densitometric scanning using a Bio-Rad (Richmond, CA) video densitometer (model 620). The Nytran membrane was then stripped of the IP-10 probe and reprobed for ß-actin mRNA. The level of ß-actin mRNA expression, which was unaffected by any of the treatments, was used as the internal control for each specimen (25).

RNase protection assay
A 297-bp rat IP-10 cDNA fragment was PCR amplified using pRLG17 plasmid DNA as a template sense primer (5'-ATGAACCCAAGTGCTGCCGTC-3') and antisense primer (5'-TTACGGAGCTCTTTTAGACCT-3'). The PCR product was subcloned into pCRII vector using the TA cloning kit (Invitrogen) and sequenced manually by the dideoxynucleotide chain termination method. The rat IP-10 antisense riboprobe (complementary RNA) was transcribed using the Maxiscript in vitro transcription kit (Ambion, Austin, TX). Before transcription, the DNA template was linearized with XbaI digestion. Transcription was carried out for 1 h at 37 C using 0.5 µg DNA template with [{alpha}-32P]CTP (ICN, Costa Mesa, CA) and SP6 polymerase. As an internal control, rat ß-actin antisense riboprobe were synthesized using pTRI-ß-actin-125-Rat template, SP6 polymerase, and the Maxiscript in vitro transcription kit (Ambion).

RNase protection assays were performed using the HybSpeed RPA kit (Ambion) as instructed by the manufacturer. Briefly, preliminary experiments were performed to determine saturating quantities of each riboprobe used in subsequent experiments. Total RNA (5–10 µg) from rat Leydig cells was resuspended in hybridization buffer containing saturating concentrations of rat IP-10 riboprobe and rat ß-actin riboprobe. The mixture was heated to 95 C for 5 min, and samples were hybridized for 1 h at 68 C. Subsequently, samples were treated with a RNase A-T1 mixture for 1 h at 37 C, precipitated, resuspended in formamide-containing gel loading buffer, and run on 5% polyacrylamide-8 M urea gels. In each gel, five [{alpha}-32P]CTP labeled RNA transcripts synthesized from Century Marker Templates (Ambion) with lengths of 100, 200, 300, 400, and 500 bases were run simultaneously with samples in a separate lane as size standards. Gels were exposed overnight and up to 3 days to Fuji RX x-ray film at -70 C with intensifying screens.

Screening of rat testis cDNA library for IP-10 cDNA
A rat testis 5'-stretch plus cDNA library was purchased from Clontech (Palo Alto, CA). The library was prepared by using mRNA from normal whole testis of an adult Sprague-Dawley. Priming method was oligo(deoxythymidine) plus random primed. The fragments were cloned into the unique EcoRI site of {lambda}gt11. Approximately 2 x 106 plaques were screened with IP-10 probes that were labeled with random primers (Life Technologies). Filters were prehybridized at 42 C in a solution containing 50% formamide for 1 h and hybridized in the same solution with the addition of 1 x 107 cpm/ml IP-10 probe at 42 C for 18 h. The filters were then washed at room temperature in 2 x SSC-0.1% SDS for 30 min and at 56 C in 0.1 x SSC-0.1% SDS for 1.5 h and exposed to Fuji RX x-ray film for 24 h at -70 C. One positive clone was obtained. The isolated phage DNA was digested with various restriction enzymes, subcloned into the pUC19 vector (Pharmacia, Piscataway, NJ), and sequenced using the Sequenase kit.

Histochemical and immunocytochemical staining of rat Leydig cells
Freshly isolated purified Leydig cells in PBS were air-dried on the 3-aminopropyltriethoxysilane (Sigma)-precoated slides. The cells were treated with 3% H2O2-methanol for 30 min at room temperature. The cells were then processed with the avidin-biotin complex method using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). The cells were incubated with the rabbit antirat IP-10 antibody (1:200 dilution) at 4 C overnight. Vectastain ABC and DAB kits (Vector Laboratories) were used for the second antibody conjugation and diaminobenzidene staining. Preimmune serum was employed in place of primary antisera to determine nonspecific immunoreactivity.

Histochemical staining for 3ßHSD enzyme activity was carried out on air-dried Leydig cells with 0.4 mM etiocholanolone as the steroid substrate as reported previously (21).

Effects of IP-10 antisense oligonucleotides on Leydig cell steroidogenesis
To evaluate the functional role of IP-10 on Leydig cells, IP-10 antisense oligonucleotides were employed. Purified adult Leydig cells (1.5 x 105 cells/ml/well) were cultured in DMEM-Ham’s F-12 with 0.1% bovine calf serum overnight. The transfection mixture consisted of 95 µl antibiotic-free OPTI-MEM I reduced serum medium (Life Technologies), 5 µl LipoTAXI reagent (Stratagene), and 25 pmol IP-10 antisense or control oligonucleotides (antisense oligonucleotides, 5'-ACGACAGCAGCACTTGGGTT-3'; control oligonucleotides, generated by scrambling the antisense oligonucleotides, 5'-TGATTAGCGCGGCAGACATC-3'), which were synthesized and HPLC purified by Oligos Etc. (Wilsonville, OR) in a polystyrene tube. Preliminary experiments indicated that 25 pmol oligonucleotides was the optimal concentration for transfection of Leydig cells. The mixture was incubated for 30 min at room temperature, and then 200 µl OPTI-MEM I medium were added. After removing the culture medium from the Leydig cell culture, the complete transfection mixture was transferred to the cells dropwise while swirling the dish. The transfection procedure was carried out under standard cell culture conditions for 4 h (5% CO2; 37 C in a humidified incubator) and stopped by replacing the transfection mixture with freshly prepared culture medium. Cultures were then treated with or without hCG (10 ng/ml) for an additional 24 h. Testosterone levels were measured in the supernatants.

Statistical analyses
All experiments were repeated at least three times. One-way ANOVA followed by Newman-Keuls multiple comparison tests were used for statistical analyses (Prism, version 2.01, GraphPad, San Diego, CA). P <= 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential display PCR compares mRNA from normal rat Leydig cells treated with and without hCG
In adult Leydig cells, LH/hCG stimulates transcription of genes for the testosterone biosynthetic enzymes, including StAR, P450scc, 3ßHSD, and P450c17 (1, 26, 27, 28). LH/hCG also increased transcription of insulin-like growth factor I receptor, IL-1, and IL-6 genes (4, 13, 29). In an attempt to identify other genes that are differentially expressed in rat Leydig cells as a result of hCG treatment, we applied the differential display technique to compare patterns of mRNA expression from primary cultured rat Leydig cells treated with and without hCG for 6 h. After screening through 20 primer combinations, dozens of differentially expressed cDNA fragments were identified. Among those, we found a cDNA fragment amplified with AP-2 and T12 MA primers that was reproducibly identified in normal Leydig cells and inhibited by hCG (Fig. 1Go). The 241-bp cDNA fragment was cloned into pCRII vector and designated pA21. The subsequent sequence analysis and GenBank database searching showed that it was the rat IP-10 gene with a high degree of homology with mob-1 cDNA (Fig. 2AGo). RT-PCR was then applied to evaluate whether IP-10 mRNA existed in normal rat Leydig cells. Sense primer (5'-ATGAACCCAAGTGCTGCCGTCATT-3') was designed from the beginning of the murine IP-10 coding sequence, and antisense primer (5'-CTACCCATTGATACATAC-3') was based on the 241-bp fragment described above. As a result, a cDNA fragment 909 bp in size was specifically amplified (Fig. 2BGo). The fragment was then cloned into the pCRII vector, and the construct was designated pRLG17. The subsequent sequence analysis demonstrated it to be the rat IP-10 gene (20). It is identical to the mob-1 cDNA sequence with the exception of five nucleotides (another two nucleotide differences were caused by upstream primer sequence based on murine IP-10) (19). Correspondingly, there is a two-amino acid difference in the coding region of the gene. Compared with the newly published rat IP-10 sequence, two nucleotides differ in the upstream primer sequence, whereas the coding sequences are identical (20).



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  		Figure 1. Differential display compares mRNA from normal rat Leydig cells treated with or without hCG (10 ng/ml) for 6 h. mRNA differential display was carried out using AP-2 and T12 MA primers. PCR products were resolved in a sequencing gel (lane 1, control; lane 2, hCG treated). The band indicated with an arrowhead shows a marked reduction in response to hCG treatment on the differential display gel.

 


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  		Figure 2. A, Nucleotide sequence of 241-bp cDNA probe identified by differential display. The flanking primers, AP2 and T12 MA, are underlined. The single C underlined is deleted in the sequence reported by Liang et al. (19 ). B, Nucleotide sequence of RT-PCR product of IP-10 cDNA. Both the start and stop codons of IP-10 protein are shown in bold. The nucleotides different from that reported by Liang et al. (19 ) are underlined and specified.

 
Tissue-specific expression of IP-10
We compared expression of the IP-10 mRNA in various adult rat normal tissues including seminiferous tubules, which account for the most of the mass in the testis. Northern blot analyses showed that IP-10 mRNA was expressed in low levels in spleen and kidney and was undetectable in heart, liver, lung, and seminiferous tubules (Fig. 3AGo). The highest expression of IP-10 mRNA was found in freshly isolated Leydig cells in the form of two transcripts, 1.5 and 2.7 kb (Fig. 3Go, A and B). The expression of both human and murine IP-10 in the absence of proinflammatory stimulation was very low or undetectable in most normal tissues (20); therefore, it is interesting that IP-10 mRNA was constitutively expressed in such high levels in Leydig cells.



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  		Figure 3. A, Northern blot analysis of total RNAs isolated from various tissues. Each lane contained 20 µg total RNAs. Lane 1, Heart; lane 2, spleen; lane 3, liver; lane 4, lung; lane 5, kidney; lane 6, seminiferous tubule; lane 7, freshly isolated Leydig cells; lane 8, Leydig cells after 24 h in culture. Hybridization was carried out with an IP-10 cDNA probe. The blot was then stripped and reprobed with ß-actin. B, Northern blot analysis of total RNA extracted from freshly isolated Leydig cells.

 
Inhibition of IP-10 gene expression by hCG
Figure 4Go shows the effects of hCG on IP-10 gene expression by RNase protection assay. Hybridization with 32P-labeled IP-10 and ß-actin complementary RNAs followed by RNase digestion and PAGE resulted in protected IP-10 and ß-actin RNA bands. The IP-10 gene was inhibited by hCG in a dose-dependent manner. The addition of 1 ng/ml hCG inhibited IP-10 mRNA levels more than 80%.



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  		Figure 4. Effects of hCG on IP-10 mRNA expression. Purified Leydig cells were cultured for 24 h. After medium change, Leydig cells were treated with 0 (lane 3), 0.1 (lane 4), 1 (lane 5), and 10 (lane 6) ng/ml hCG for 6 h. Total RNAs were extracted for RNase protection assay. Lanes 1 and 2 show rat IP-10 and rat ß-actin riboprobes that were carried through the assay in the presence of only nontarget yeast RNA and were subsequently incubated without and with RNase, respectively. Probes in lane 1 were diluted before loading. Lane 7 shows a molecular mass marker. Lanes 1' and 7' are from the same blot with lighter exposure. Similar results were obtained in two other separate experiments. A, A representative blot; B, IP-10/actin ratio.

 
Activation of IP-10 expression in primary cultured rat Leydig cells by mIL-1{alpha}, mTNF{alpha}, and mIFN{gamma}
We compared the expression of IP-10 mRNA in primary cultured rat Leydig cells treated with and without mIL-1{alpha}, mTNF{alpha}, and mIFN{gamma}. RNase protection assay analyses demonstrated that the expression IP-10 mRNA in Leydig cells was increased significantly in response to these cytokines. mIL-1{alpha}, mTNF{alpha}, and mIFN{gamma} increased IP-10 mRNA levels by 3.3-, 10-, and 26-fold, respectively. Concomitant addition of mIL-1{alpha}, mTNF{alpha}, and mIFN{gamma} synergistically increased the IP-10 mRNA level by 58-fold (Fig. 5Go).



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  		Figure 5. Effects of mIL-1-{alpha}, mTNF{alpha}, and mIFN{gamma} on IP-10 mRNA expression. Purified Leydig cells were cultured for 24 h. After medium change, Leydig cells were treated with mIL-1{alpha} (10 ng/ml; lane 4), mTNF{alpha} (10 ng/ml; lane 5), mIFN{gamma} (500 U/ml; lane 6), or in combination (mIL-1{alpha}, mTNF{alpha}, and mIFN{gamma}; lane 7) for 6 h. Lane 3 is the basal control without any treatment. Total RNAs were extracted for RNase protection assay. Lanes 1 and 2 show rat IP-10 and rat ß-actin riboprobes that were carried through the assay in the presence of only nontarget yeast RNA and were subsequently incubated without and with RNase, respectively. Probes in lane 1 were diluted before loading. Lane 8 shows a molecular mass marker. Similar results were obtained in two other separate experiments. I, mIL-1{alpha}; T, mTNF{alpha}; IFN, mIFN{gamma}.

 
Screening of rat testis cDNA library for IP-10 cDNA sequence
Interested by the two mRNA transcripts of IP-10 found in isolated rat Leydig cells, we used IP-10 cDNA as a probe to screen a rat testis cDNA library. As Northern blot results indicated that IP-10 was expressed in normal Leydig cells but not in seminiferous tubules, IP-10 cDNA should be rare in the library because Leydig cells account for only a small percentage of the whole testes. A positive plaque was identified after screening 2 x 106 plaques. The 1.7-kb cDNA insert was excised by EcoRI and cloned into the pUC 19 vector, with the resultant construct termed pRLIP10. Subsequent sequence analysis revealed that the insert consisted of a partial IP-10 structural gene sequence as well as an intron (Fig. 6Go). One explanation for the incompleteness of this cDNA sequence is that the EcoRI site in the large full-length cDNA sequence was not protected by methylation, an important step in library construction. It may thus have been cleaved by EcoRI digestion before ligation into the EcoRI cloning site of phage DNA. The sequences shared between this large but incomplete cDNA and the smaller published IP-10 cDNA are identical. When we compared the sequence of this 1.7-kb fragment with the genomic sequence of murine IP-10, we found a high level of homology between these two sequences. A computer search revealed a large number of potential regulatory elements in the 5'-flanking region of the rat IP-10 gene, which include TATA, CAAT, AP-1, nuclear factor-{kappa}B1 (NF-{kappa}B1), NF-{kappa}B2 and IFN stimulus response element (ISRE).



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  		Figure 6. Nucleotide sequence of a 1.7-kb EcoRI fragment cloned from screening of rat testis cDNA library using IP-10 cDNA as a probe.

 
Histochemical and immunocytochemical staining of purified Leydig cells
As shown in Fig. 7aGo, purified Leydig cells stained strongly positive for 3ßHSD. Figure 7bGo shows that purified Leydig cells also stained positively for IP-10. This unequivocally confirms that IP-10 is indeed present in Leydig cells. Figure 7cGo shows negative control for IP-10 immunostaining.



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  		Figure 7. Histochemical and immunocytochemical staining of purified Leydig cells were carried out as described in Materials and Methods. a, 3ßHSD staining of purified Leydig cells. Magnification, x100. b, IP-10 staining of purified Leydig cells. Magnification, x100. c, IP-10 staining of purified Leydig cells, negative control. Magnification, x100.

 
Effects of IP-10 on Leydig cell function
When purified mature Leydig cells were transfected with IP-10 antisense oligonucleotides, both basal and hCG-stimulated testosterone formation were enhanced (Table 1Go). Testosterone formation in cells transfected with control oligonucleotides was comparable to that in untransfected cells, indicating that control oligonucleotides were not toxic to Leydig cells.


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  		Table 1. Effects of IP-10 antisense oligonucleotides on Leydig cell steroidogenesis

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have identified a gene that is differentially expressed in normal adult rat Leydig cells, and whose expression is inhibited by hCG but stimulated by mIFN{gamma}. Furthermore, mIL-1{alpha}, mTNF{alpha}, and mIFN{gamma} have synergistic effects on the induction of this gene. DNA sequence analysis identified this gene as rat IP-10. Histochemical and immunocytochemical staining revealed that purified Leydig cells stained positively for 3ßHSD and IP-10. This confirms that IP-10 is indeed present in rat Leydig cells. IP-10 antisense oligonucleotides enhanced both basal and hCG-induced testosterone formation. This suggests that endogenous IP-10 has an inhibitory effect on Leydig cell steroidogenesis.

In recent years, various cytokines have been identified in the testis that may have paracrine or autocrine effects on testicular function. An IL-1-like activity was identified in the testis, seminiferous tubule-conditioned medium, and adult rat testis interstitial fluid (30, 31, 32, 33). Purified adult rat Leydig cells express both IL-1{alpha} and IL-1ß mRNAs, with levels of IL-1ß mRNA significantly higher than those of IL-1{alpha} mRNA (3, 4). Furthermore, IL-1ß mRNA in Leydig cells increase markedly in intact rats after hCG treatment (4). IL-1 affects Leydig cell steroidogenesis and proliferation, and spermatogenesis (10, 11, 12). IL-6 is secreted by purified adult Leydig cell preparation, and its release is increased by hCG and IL-1ß (13). Cultured mouse Sertoli cells also produce IL-6, and its production is enhanced by IL-1{alpha}, IL-1ß, TNF{alpha}, and lipopolysaccharide, but inhibited by IFN{gamma} (34). IFN{gamma} protein and corresponding mRNA are expressed by peritubular, Sertoli, and germ cells (35). Both IFN{gamma} mRNA and protein are found in early spermatids (35). Transgenic mice carrying extra mouse IFN genes are sterile (36, 37). In the present study, we have identified yet another chemokine, IP-10, which is expressed in high levels in Leydig cells. As IP-10 antisense oligonucleotides enhanced basal and hCG-induced testosterone formation, endogenous IP-10 may have an inhibitory effect on Leydig cell steroidogenesis.

IP-10 was first identified as an inflammatory protein with a molecular mass of about 10 kDa in the IFN{gamma}-treated human U937 histiocytic lymphoma cell line (15). IP-10 mRNA expression is also induced in human mononuclear cells, osteoblasts, endothelial cells, keratinocytes, and fibroblasts by IFN, platelet-derived growth factor, IL-1ß, and TNF{alpha} (15, 16, 38, 39, 40, 41). IP-10 has been cloned from humans, mice, and rats (15, 16, 19, 20). Based upon structural similarities, IP-10 has been categorized as a member of the -C-X-C- chemokine family (17, 18). IP-10 has either mitogenic or antitumor effects depending on the cell system studied (20, 42, 43, 44, 45). The mob-1 gene was originally reported by Liang et al. (19) in an effort to identify genes that are transcriptionally regulated during cell transformation caused by the cooperation of the ras oncogene and p53 tumor suppressor gene. mob-1 is the homolog of rat IP-10 (20). Oncogenic Ras as well as serum growth factors that activate endogenous Ras can induce mob-1 expression. Transfection assays established that mob-1 is a downstream target gene of the Ras signaling pathway, and oncogenic mutation in H-ras results in constitutive expression of the gene (19). As IP-10 is a potent chemokine, it is attractive to speculate that it may be involved in both acute and chronic inflammation and the pathogenesis of autoimmune disorders of the testis.

Regulation of IP-10 gene expression has been reported in various cell types. In a mouse macrophage-like cell line, IP-10/crg-2 was induced by IFN{gamma}, but not by TNF{alpha} or IL-1 (16). In rat vascular smooth muscle cells, IL-1ß or TNF{alpha} also had no significant effect on IP-10 mRNA expression. However, the combination of IFN{gamma} with IL-1ß or TNF{alpha} had a synergistic effect on IP-10 induction (20). IP-10 mRNA expression has also been investigated in cell lines of mesenchymal origin (39). In BALB/3T3 fibroblast cells, IFN{gamma}, TNF{alpha}, and IL-1{alpha} markedly induced IP-10 mRNA expression, whereas only TNF{alpha} induced significant expression of IP-10 mRNA in MC-3T3-E1 osteoblastic cells (39). In the present study, we found that IFN{gamma} is more potent than either IL-1 or TNF{alpha} in inducing IP-10 expression. Furthermore, IL-1, TNF{alpha}, and IFN{gamma} had a synergistic effect on the induction of IP-10 expression in rat Leydig cells. Ohmori and Hamilton reported that IFN{gamma}-induced transcriptional activation of the IP-10 gene in a macrophage cell line is mediated by regulatory sequences found in the region immediately upstream of the transcription start site, including an ISRE and two {kappa}B sites (46, 47). In the present study, we have also identified a large number of potential regulatory elements in the 5'-flanking region of rat IP-10 gene, including TATA, CAAT, AP-1, NF-{kappa}B1, NF-{kappa}B2, and ISRE.

The mechanism of action of IP-10 is believed to be mediated by its binding to cell surface heparan sulfate proteoglycans (48). Platelet factor-4, but not IL-8, monocyte chemoattractant protein-1, RANTES (regulated upon activation, normal T cell expressed and secreted), or monocyte inflammatory protein-1{alpha} or -1ß, can compete effectively with IP-10 for binding to the cell surface (48). IP-10 further shares with platelet factor-4 the ability to inhibit endothelial cell proliferation (48). Very recently, a human receptor that is specific for IP-10 was cloned and characterized from CD4+ T cells (49). The receptor cDNA has an open reading frame of 1104 bp, encoding a protein of 368 amino acids with a molecular mass of approximately 40 kDa. The sequence includes seven putative transmembrane segments characteristic of G protein-coupled receptors. The gene for the receptor, designated CXCR3, is highly expressed in IL-2-activated T lymphocytes, but is not detectable in resting T lymphocytes, B lymphocytes, monocytes, or granulocytes (49). Cells transfected with the receptor cDNA demonstrated calcium fluxes and chemotaxis in response to Mig (monokine induced by IFN-{gamma}) and IP-10, but not to other chemokines (49).

In conclusion, we have identified a chemokine, IP-10, that is expressed in high levels in freshly isolated and primary cultured rat Leydig cells. Expression of IP-10 is induced by IFN{gamma}, TNF{alpha}, and IL-1{alpha}, but is inhibited by hCG. IP-10 may be involved in the paracrine and autocrine regulation of testicular function. Furthermore, as IP-10 is a potent chemokine, it may be involved in the acute and chronic inflammatory disorders of the testis.


   Footnotes
 
1 This work was supported by the Department of Veterans Affairs Medical Research Fund (to T.L.). Back

Received January 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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