Hum. Reprod. Advance Access originally published online on September 22, 2006
Human Reproduction 2006 21(11):2801-2809; doi:10.1093/humrep/del256
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The high mobility group box chromosomal protein 1 is expressed in the human and rat testis where it may function as an antibacterial factor
Department of Woman and Child Health, Astrid Lindgren Children’s Hospital, Pediatric Endocrinology Unit, Karolinska Hospital, Stockholm, Sweden
1 To whom correspondence should be addressed at: Department of Woman and Child Health, Pediatric Endocrinology Unit, Karolinska Institute and University Hospital (Q2:08), SE-171 76 Stockholm, Sweden. E-mail: mona-lisa.strand{at}ki.se
* Previously Cecilia K.Jonsson
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Abstract |
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BACKGROUND: The high mobility group box chromosomal protein 1 (HMGB1) was originally shown to be a nuclear DNA-binding protein that activates transcription and promotes differentiation. More recently, there have been reports that HMGB1 may also function as a pro-inflammatory and antibacterial factor. The aim of this study was to investigate the testicular expression and antibacterial functions of HMGB1 to elucidate a possible role of HMGB1 in the testicular barrier defence against infections. METHODS AND RESULTS: RT–PCR and in situ hybridization revealed high-level testicular expression of HMGB1 mRNA and localization of this expression to the Sertoli cells and germ cells of the human and rat testis. In addition, immunohistochemical examination demonstrated the presence of the corresponding protein in Sertoli cells and spermatogonia in human and rat testes. Western blotting detected abundant amounts of the HMGB1 protein in the interstitial and intratubular fluids of the intact adult rat testis. Finally, the HMGB1 protein purified from both human and rat testis by reversed-phase high-performance liquid chromatography (HPLC) exerted antibacterial activity towards Bacillus megaterium in an inhibition zone assay. CONCLUSION: HMGB1 is expressed by Sertoli cells and germ cells in the mammalian testis. In addition, purified testicular HMGB1 shows antibacterial activity, indicating that this protein may function as a paracrine host defence factor in the testis.
Key words: anti-microbial defence/cytokines/HMGB1/Sertoli cells/testis
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Introduction |
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The family of high mobility group box of chromosomal (HMGB) proteins presently contains three different members, designated HMGB1-3 and originally named for their high electrophoretic mobility in polyacrylamide gels (Johns, 1982
). The HMGBs consist of three different domains, that is, two DNA-binding domains (HMG boxes A and B) and a negatively charged C-terminal domain. HMGB2 is expressed in many different tissues during embryogenesis but is detected only in lymphoid organs and the testis in the adult mouse (Ronfani et al., 2001). Hmgb2-null mutant mice are viable, but the males exhibit reduced fertility because of degeneration of Sertoli and germ cells and the immobility of their spermatozoa (Ronfani et al., 2001). HMGB3 is expressed only during embryonic development (Vaccari et al., 1998). In contrast, HMGB1 is expressed by a wide variety of mammalian cells and, until recently, was regarded primarily as a nuclear protein capable of binding with high affinity to certain types of DNA structures (Bianchi, 1988; Bianchi et al., 1989; Maher and Nathans, 1996; Murphy et al., 1999). Lately, however, HMGB1 has been shown to also bind to certain transcription factors (Bianchi and Beltrame, 2000; Brickman et al., 1999), as well as to promote both neurite outgrowth and tumour growth through interaction with the receptor for advanced glycation end-products (RAGE) (Huttunen et al., 1999; Taguchi et al., 2000). Furthermore, several novel extracellular functions carried out by HMGB1 have been described recently (Wang et al., 2004). This protein acts as a late mediator of endotoxin lethality and is present at elevated levels in the serum of patients with sepsis (Wang et al., 1999). Moreover, HMGB1 potently stimulates cytokine release by human monocytes (Andersson et al., 2000) and, in one study, was found to cause pneumonic inflammation (Abraham et al., 2000). HMGB1 is also present in the synovium of arthritic human joints where it constitutes a potential target for the therapeutic treatment of rheumatoid arthritis (Andersson and Erlandsson-Harris, 2004). In addition to RAGE, extracellular HMGB1 binds to several other surface receptors, including Toll-like receptor (TLR) 2 and TLR 4, thereby eliciting several various immunostimulatory and chemotactic responses (Andersson et al., 2000; Degryse et al., 2001; Stern et al., 2002; Park et al., 2004). Thus, HMGB1 is a pleiotropic differentiation factor and a mediator of host defences, which acts specifically on a wide range of target cells (Lotze and Tracey, 2005). Recently, we discovered that this same protein is produced by the human adenoid gland and exerts potent antibiotic activity, killing bacteria within a time frame of seconds or minutes (Zetterstrom et al., 2002). Although epididymitis is relatively common, bacterial infection of the urogenital tract that ascends to the testis occurs rarely, and when it does, may damage the germinal epithelium and impair fertility. Local mechanisms for protecting the developing germ cells from such infections are therefore of considerable importance, and several systems of this kind have been described previously (Agerberth et al., 1995; Dejucq et al., 1997; Grandjean et al., 1997; Nieschlag and Behre, 2001; Com et al., 2003; Maxwell et al., 2003; Semple et al., 2003). Furthermore, the testis constitutively expresses several immunoregulatory cytokines (Strand et al., 2005; Sultana et al., 2000; Sultana et al., 2004) and represents a unique immunological environment that allows prolonged survival of allografts and frequent relapse of acute lymphocytic leukaemia in boys (Ritzen, 1990; Nasr et al., 2005). We report here that both the HMGB1 protein and corresponding mRNA are expressed at high levels by Sertoli cells in the human as well as in the rat testis and, in addition, that the purified protein exhibits antibacterial activity. |
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Tissue samples and animals
Testis biopsies were obtained from men undergoing examination for infertility. All of the materials employed for RT–PCR in this study originated from three men, 30–40 years old, who later gave rise to normal pregnancies and in whom spermatogenesis demonstrated qualitatively and quantitatively normal histology. The testis that was used for the in situ hybridization was obtained from a 70-year-old man with prostate cancer, and the material used for the immunohistochemistry was obtained from the healthy testis of a 33-year-old man with seminoma. In the case of the animal experiments, male Sprague–Dawley rats (B & K Laboratories, Sollentuna, Sweden), 10 and 60 days of age, were sacrificed by CO2 inhalation and samples of their tissues rapidly frozen thereafter to –80°C. The experimentation was approved by the Northern Stockholm Animal Ethics Committee (Reg. No. N151/01; N 218/04) and the studies on human testis biopsies by the local ethics committee at Huddinge University Hospital (no. 325/95).
RT–PCR
Total RNA was extracted from snap-frozen tissue samples with the Ultraspec II Kit (BIOTECX Laboratories, Houston, TX, USA) following the protocol provided by the manufacturer. Primer pairs specific for amplification of rat and human HMGB-1, as well as the two internal standards rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and human 
-actin, were designed on the basis of published nucleotide sequences (Table I). The Superscript cDNA kit (Life Technologies, Inc., Gaithersburg, MD, USA) and RNase Inhibitor (Roche Diagnostics Corp., Mannheim, Germany) were then utilized to synthesize cDNA. The end-products thus obtained were subjected directly to PCR in an Perkin-Elmer GeneAmp PCR System 2400, in the presence of 25 pmol primers, 10 pmol dNTP, 75 pmol High-Fidelity PCR buffer and 1.75 U Expand High-Fidelity DNA Polymerase (Roche Diagnostics GmbH). Thereafter, the resulting reaction products were analysed by gel electrophoresis on 2% agarose gels containing ethidium bromide (15 µg per 100 ml gel). Unstimulated human monocytes and human adenoid tissue, together with a crude source of activated macrophages obtained from inflammation-induced peritoneal exudates, collected by peritoneal lavage of a 60-day-old rat, 2 days after i.p. injection of 3 ml of Freund’s complete adjuvant (Jonsson et al., 1999) were employed as positive controls. In the case of the negative controls, no RNA or cDNA was added to the RT–PCR system. Duplicate PCR amplifications of HMGB1 transcripts in the three human samples was performed twice, followed by semi-quantification employing Image J (http://rsb.info.nih.gov/ij/), using -actin as a reference gene.
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Histochemical examination by in-situ hybridization histochemistry
Sections of tissue samples (10 µm thick) were thaw-mounted onto slides (ProbeOn, Fisher Scientific, Pittsburgh, PA, USA) and in-situ hybridization histochemistry (ISHH) was performed according to Dagerlind et al. (1992)
, employing an HMGB1-specific, 50 bp long oligonucleotide probe (DNA Technology, Aarhus, Denmark) with the following sequence: 5'-TCTTCTTCCTCCTCTTCCTCATCCTCT- TCATCCTCCTCGTCGTCTTCCTC-3'. A random probe (Jonsson et al., 1999), with no similarity to known gene sequences (GenBank, NIH), was utilized as a negative control. Briefly, the sections were hybridized with these probes for 18 h at 42°C in humidified boxes and thereafter rinsed five times for 15 min each time in saline sodium citrate (SSC) buffer (150 mM NaCl, 15 mM sodium citrate, pH 7.0) at 60°C. Subsequently, the tissue sections were dehydrated, air-dried, dipped in photographic emulsion (NTB-2, Kodak, Rochester, NY, USA), exposed for 3–4 weeks at –20°C, developed, counterstained with Carmalume and mounted (Entellan, VWR International, Stockholm, Sweden). Finally, these preparations were examined using bright- and dark-field microscopy (Nikon Eclipse, E800).
Immunohistochemistry
Immunohistochemical staining was performed as described by R & D Systems, UK. The primary antibody was raised by immunizing rabbits with a peptide corresponding to amino acid residues 165–183 of rat/mouse HMGB1 (Innovagen AB, IDEON Research Park, Lund, Sweden), affinity purified and used at a concentration of 2 µg/ml. The secondary antibody was a fluorescein isothiocyanate (FITC)-conjugated affinity-purified F(ab') fragment donkey anti-rabbit immunoglobulin G (IgG) (H+L) antibody (Jackson ImmunoResearch Laboratories, West Growe, PA, USA), diluted to a final concentration of 30 µg/ml. Some sections were double stained for HMGB1 and ED2, a rat macrophage-specific antigen, as described previously (Jonsson et al., 2001). Slides were mounted with fluorescent mounting medium (Dako Corporation, Carpinteria, CA, USA) and examined by fluorescence microscopy (Nikon Eclipse, E800, Bergström Instrument, Solna, Sweden).
Double immunohistochemistry
For double immunostaining of HMGB1 and transferrin and for co-staining with anti-HMGB1 antibodies and 4,6-diamidine-2-phenylindole, dihydrochloride (DAPI), we fixed the testes from 60-day-old rats in Bouin’s solution, dehydrated them through graded series of alcohol and embedded them in paraffin wax. Five-micrometre paraffin sections were prepared from these testes, followed by incubation in xylene, two times for 10 min each time, and subsequently rehydrated through graded series of alcohol. Five-micrometre paraffin sections from a human testis were also treated in the same way. The sections were then incubated with antigen retrieval solution (10 mM sodium citrate buffer, pH 6.0) for 10 min at 95°C, followed by washing in Tris-buffered saline containing 0.05% Tween 20 (TBST), 5 min each time. The sections were then saturated with TBST containing 1% bovine serum albumin (BSA) for 1 h at room temperature, before incubation for overnight at 4°C with 2 µg/ml of the rabbit polyclonal anti-HMGB1 antibodies (Innovagen AB, IDEON Research Park), diluted in TBST containing 1% BSA. These slides were next washed three times for 5 min each time in TBST, followed by incubation for 1 h at room temperature with Cy3-conjugated affinity-purified F(ab') fragment donkey anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) at a final concentration of 15 µg/ml. After washing three times for 5 min each time in TBST, the sections were saturated once again with TBST containing 1% BSA, washed and incubated with goat polyclonal anti-transferrin antibodies (sc-22597, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), diluted to a final concentration of 1 µg/ml in TBST containing 1% BSA, for overnight at 4°C. These slides were then washed in TBST, incubated for 1 h at room temperature with FITC-conjugated affinity-purified donkey anti-goat IgG (H+L) (Jackson ImmunoResearch Laboratories), diluted to a final concentration of 30 µg/ml in TBST containing 1% BSA. Following incubation, the sections were washed in TBST and mounted with Vectashield® Hard SetTM mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). Finally, the sections were examined by fluorescence microscopy (Nikon Eclipse, E800, Bergström Instrument). Sections incubated with only the secondary antibodies or with a species and isotype matched irrelevant antibody (Calbiochem, EMD Biosciences Inc., Darmstadt, Germany) were used as negative controls.
Protein purification of human and rat HMGB1 by reversed-phase high-performance liquid chromatography
Frozen tissue samples from human and rat testes were homogenized in and extracted overnight with 60% aqueous acetonitrile containing 1% trifluoracetic acid (TFA) (Moore et al., 1991), following which the extracts thus obtained were centrifuged at 13,000 g for 15 min to remove debris and the resulting supernatants lyophilized and the residues obtained dissolved in water. These preparations were then subjected to reversed-phase high-performance liquid chromatography (RP-HPLC), employing gradient elution with acetonitrile from a µRPC C2/C18 SC 2.1/10 column (product no. 17-0704-01, Pharmacia Biotech, Uppsala, Sweden), utilizing a micropurification system (SMART System; Pharmacia Biotech). Solvent A was aqueous 0.17% TFA and solvent B acetonitrile containing 0.15% TFA. Protein separation was accomplished in two steps, with a flow rate of 200 µl/min and initial isocratic elution with 0.5 ml 100% A in both cases. Subsequently, the first step involved sequential elution with 5 ml 0–100% B (all gradients were linear), 1 ml 100% B and 1 ml 100–0% B). Two hundred-microlitre fractions were collected throughout this entire run and lyophilized, following which the residues obtained were dissolved in water and tested for the presence of HMGB1 using a dot immunoblot assay (see below). This assay revealed that HMGB1 was eluted from the column with 25–50% B, which is also the case in connection with preparation of the HMGB1 protein from human adenoid tissue (Zetterstrom et al., 2002). Accordingly, these fractions from the first run were subjected to a second purification step involving sequential elution with 0.5 ml 0–25% B, 2.5 ml 25–50% B, 1 ml 50–100% B, 0.5 ml100% B and 1 ml 100–0% B. In this case, 100-µl fractions were collected 170 during the elution with 25–50% B and dot blotted for HMGB1.
Dot immunoblotting for HMGB1
Two microlitres of each chromatographic fraction were dotted onto to a Hybond nitrocellulose membrane (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK), pre-blocked with 1% BSA in Tris-buffered saline containing 0.1% Tween 20 and then incubated with the primary rabbit anti-rat HMGB1 antibodies, described above. HMGB1-antibody complexes were visualized employing a horse-radish peroxidase-conjugated donkey anti-rabbit antibody, diluted 1:5000 (Amersham Pharmacia Biotech) and developed using the ECL system (Amersham Pharmacia Biotech) according to the manufacturer’s recommendations.
Antibacterial assay
The HMGB1-positive and HMGB1-negative fractions obtained by the reversed-phase HPLC were pooled in separate tubes, lyophilized and dissolved in water at higher (1:1) and lower (1:2) concentrations before they were analysed for their antibacterial activity. Bactericidal activity against Bacillus megaterium (Bm11) was evaluated by performing an inhibition zone assay on thin (1-mm thick) agar plates (Hultmark et al., 1983), containing Luria-Bertani broth (LB), 1% agarose and approximately 104–105 bacteria undergoing log phase growth. Small wells (3 mm in diameter) were punched in these plates and loaded with 3 µl of the pooled fractions described above. After incubation overnight at 30°C, the diameters of the zones of growth inhibition were measured as reflections of the ability of the purified human and rat HMGB1 protein to kill these bacteria. As a positive control and reference substance, LL-37 (from FALL-39), a human antibacterial peptide (Agerberth et al., 1995; Gudmundsson et al., 1996) synthesized by Innovagen AB (IDEON Research Park), was used. Escherichia coli-derived recombinant rat HMGB1 protein were not included as a positive control in this experiment but have previously been shown to posses antibacterial activity (Zetterstrom et al., 2002).
Extraction of protein from rat testicular tissue
One hundred and fifty milligrams of testicular tissue samples from 60-day-old rats was homogenized in 2 ml 62.5 mM Tris–Cl (pH 6.8)—10% glycerol—2% sodium dodecyl sulphate (SDS). The resulting tissue extracts were then centrifuged for 5 min at 10,000 g and the supernatants obtained collected and assayed for the total protein content with the Bio-Rad protein assay (Bio-Rad Laboratories, Herkules, CA, USA) according to the manufacturer’s instructions.
Collection of interstitial fluid and seminiferous tubular fluid from rat testis
Interstitial fluid was collected by centrifugation of the decapsulated, but otherwise intact, testis in a centrifuge containing a 70-µm cell restrainer (Becton Dickinson, Lincoln Park, NJ, USA) for 5 min at 300 g. To collect tubular fluid, we first dissected out long segments of randomly selected seminiferous tubules, rinsed these in phosphate-buffered saline (PBS) to remove interstitial fluid and then forced these segments through a 21-gauge needle (Setchell et al., 2002). Subsequent centrifugation of the samples at 10,000 g for 15 min provided supernatants containing the tubular fluid. To evaluate leakage of intracellular proteins during these preparation procedures, we determined the level of the intracellular protein caspase-3 in the interstitial and tubular fluid samples obtained, as described previously (Strand et al., 2005).
SDS-PAGE/western blotting
A total protein of 12.5 µg extracted from testicular tissue or in tubular or interstitial fluid or 1.5 µl of the human or rat HMGB1 fractions purified by reversed-phase HPLC (see Protein purification of human and rat HMGB1 by reversed-phase HPLC) was diluted in reducing SDS loading buffer and then applied to a 15% polyacrylamide gel (Criterion, Bio-Rad Laboratories). In connection with the subsequent electrophoresis, prestained Broad Range SDS–polyacrylamide gel electrophoresis (PAGE) standards (Bio-Rad Laboratories) were used as size markers. The protein bands thus obtained were transferred to Hybond-P polyvinylidene diflouride membranes (Amersham Pharmacia Biotech) in a buffer containing 25 mM Tris and 185 mM glycine, pH 8.3 together with 20% methanol. Following this transfer, the membranes were blocked for 1 h in TBS containing 0.1% Tween 20, supplemented with 5% non-fat dry milk and then washed three times with TBS containing 0.1% Tween 20. Thereafter, these membranes were incubated overnight at 4°C with affinity-purified rabbit anti-rat HMGB1 antibodies (See Immunohistochemistry; diluted 1:8000) or with rabbit anti-caspase-3 antibodies (sc-7148, reactive towards human, rat and mouse proteins, Santa Cruz Biotechnology; diluted 1:2500) in blocking buffer. After washing, the membranes were incubated with horse-radish peroxidase-conjugated donkey anti-rabbit antibodies (Amersham Pharmacia Biotech; diluted 1:5000), and the immunoblots then developed with the ECL Plus western blotting agent (Amersham Pharmacia Biotech) and exposed to X-ray film (Amersham Pharmacia Biotech) for visualization of HMGB1 and caspase-3. Recombinant rat HMGB1 protein, produced in E. coli and tagged with calmodulin (Wang et al., 1999), which results in a slightly higher molecular weight compared with native HMGB1, was utilized as a positive control.
Statistical analysis
One-way analysis of variance (ANOVA) or Kruskal–Wallis one-way ANOVA on ranks was performed on duplicate samples from the RT–PCR amplifications of HMGB1 in the three human testis extracts. Two independent experiments were performed. P < 0.05 was considered statistical significant.
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Employing RT–PCR, we clearly revealed the presence of HMGB1 mRNA in the adult human and rat testis, as well as in the testis of the prepubertal 10-day-old rat (Figure 1A and B). Semi-quantification of the HMGB1 mRNA expression showed that there was no variation in the HMGB1 expression levels in the three human testes. Furthermore, in situ hybridization with an HMGB1-specific probe demonstrated that the HMGB1 mRNA in the human adult testis showed a localization to the seminiferous tubules that corresponded well to that of the Sertoli cells (Figure 2A) and possibly also to germ cells with no apparent expression in the interstitial space or blood vessels. In control sections of human adult testis hybridized to a random probe, no specific hybridization signal was detected (Figure 2B). In case of the prepubertal 10-day-old rat, potent expression of HMGB1 mRNA was observed in the central portions of the seminiferous tubules (Figure 2C), corresponding to the cytoplasm of immature Sertoli cells at this developmental stage. Weak expression was also apparent in the interstitial space, whereas hybridization with a random probe resulted in no specific signal (Figure 2D). In the adult 60-day-old rat testis, HMGB1 mRNA was also potently expressed in the seminiferous tubules (Figure 2E), with columnar pattern that again corresponded well to the localization of Sertoli cells, although expression in germ cells is also possible. There was no apparent variation in the level of this expression in different stages of the seminiferous tubules. The presence of a weak hybridization signal originating from certain cells in the interstitial compartment could not be excluded, although the cell types involved could not be determined. Again, negative-control sections did not display any localized hybridization signal (Figure 2F). Immunohistochemistry employing affinity-purified antibodies directed specifically towards HMGB1 was used to localize the expression of this protein in the adult rat testis. The potent immunoreactivity exhibited by the seminiferous tubules had a localization corresponding to Sertoli cells (Figure 2G) and spermatogonia. In addition, HMGB1 protein expression was evident in certain interstitial cells, which may correspond to testicular macrophages because these cells also were positive for ED2 (not shown). In human testis, the HMGB1 protein was found in the nuclei of germ cells, Sertoli cells, peritubular myoid cells and endothelial cells as well as in the cytoplasm of Sertoli cells (Figure 2H). The nuclear and cytoplasmic localization of HMGB1 in the human testis was also confirmed by co-staining with DAPI (not shown). Control sections in which the primary antibody was excluded from the incubation mixture and sections that were incubated with a species and isotype-matched irrelevant antibody (not shown) demonstrated no staining whatsoever. To further investigate the tubular expression of HMGB1 and to confirm the presence of HMGB1 in Sertoli cells, we performed double immunostaining for HMGB1 and transferrin on paraffin sections from testes from 60-day-old rats. Partial co-localization of transferrin and HMGB1 was observed in these sections, indicating that HMGB1 is present in the Sertoli cell cytoplasm (Figure 3A, B and D, arrows). However, in some seminiferous tubules, the HMGB1 protein was strictly located to the nucleus of Sertoli cells and spermatogonia (Figure 3A, B and C, arrowheads). No HMGB1 staining was seen in the luminal compartment of the seminiferous tubules. To investigate further, whether HMGB1 is secreted into the interstitial and/or intraluminal compartments of the testis, we collected interstitial and intratubular fluids from the testes of adult rats and subjected these fluids to western blotting. HMGB1 was present both in interstitial and intratubular fluid (Figure 4). However, there was detectible leakage of the intracellular protein caspase-3 into this intratubular fluid, although none of this marker protein was present in the interstitial fluid (not shown). Finally, to determine whether testicular HMGB1 possesses antibacterial activity, we extracted this protein from the adult human and rat testis and purified it by HPLC. Chromatographic fractions from the first run were lyophilized and analysed by dot immunoblot employing an antibody directed specifically against HMGB1. The fractions demonstrating immunoreactivity were subsequently re-chromatographed (Figure 5), and the new fractions obtained subjected to both the dot immunoblot assay and western blotting (Figures 4 and 5). In the case of the human and rat testis, fractions 12–15 (Figure 5A) and fractions 12–16 (Figure 5B), respectively, were the fractions that contained HMGB1 immunoreactivity. Both the HMGB1-positive and the HMGB1-negative fractions from this second step were pooled in different tubes, lyophilized, dissolved in water and analysed for their antibacterial activity at two concentrations. Only the fractions that contained human and rat testicular HMGB1 inhibited the growth of the test bacterium B. megaterium (Figure 6). Run in parallel, the human antibacterial peptide LL-37 (used as a positive control) and water (negative control) gave the expected results (Figure 6).
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These findings reveal that both HMGB1 mRNA and protein are expressed at high levels in the human and rat testis, with a localization to Sertoli cells and germ cells. We have also demonstrated that the HMGB1 protein purified from the human and rat testis exerts antibacterial activity. Even though that the urogenital tract is a common locus for bacterial infections, ascensions of such infections to the testis occurs only rarely. One explanation for this might be the presence of local antibacterial factors in the testis, including HMGB1, as shown here, and LL-37 (Agerberth et al., 1995
; Gudmundsson et al., 1996). These factors may be present at very high local concentrations and constitute part of a tissue surveillance system designed to protect developing germ cells from infections and other harmful agents. Indeed, antimicrobial peptides belonging to the defensin group have been shown to be produced by Sertoli and Leydig cells in the mouse testis (Grandjean et al., 1997). Defensins exhibit antimicrobial activity against a wide range of bacteria, fungi, protozoa and viruses (Lehrer et al., 1993), and recently, the existence of a testicular antiviral defence system has also been described (Dejucq et al., 1997). The possibility that HMGB1 may also be active against micro-organisms other than bacteria remains to be examined. Our immunohistochemical analyses revealed high-level of HMGB1 expression in the basal compartment of the rat and human seminiferous tubules. This is consistent with previous results, demonstrating that HMGB1 is expressed in spermatogonia in the mouse testis (Muller et al., 2004). However, we observed co-localization of the HMGB1 and transferrin expression in certain tubules in the rat testis, which shows that HMGB1 is also expressed in the cytoplasm of Sertoli cells. We also found cytoplasmic and nuclear expression of HMGB1 in the Sertoli cells of the human testis, in addition to nuclear expression in germ cells, peritubular myoid cells and endothelial cells. The localization of HMGB1 expression to Sertoli cells is of considerable interest because this cell type has many specialized functions centred around the well-being of the developing germ cells, such as creating the ‘Sertoli cell barrier’ that separates the post-meiotic germ cells from the interstitial compartment and acting as ‘nurse’ cells (Russell and Griswold, 1993). We have shown previously that interleukin-1
(IL-1) is expressed by Sertoli cells in the adult rat testis in a stage-dependent fashion (Jonsson et al., 1999). The expression of HMGB1 by Sertoli cells, reported here, demonstrated no apparent stage dependency and was also evident in the prepubertal testis in contrast to the expression of IL-1. However, the intracellular localization of HMGB1 varied between different seminiferous tubules and also within a single tubuli, being either nuclear or cytoplasmic. These results are in agreement with a previous report, demonstrating high levels of HMGB1 both in the nuclear and in the cytosolic fractions of homogenized testis tissue (Mosevitsky et al., 1989). The cytoplasmic localization of HMGB1 may suggest that HMGB1 plays extra nuclear roles in the testis and that Sertoli cells may even secrete HMGB1 to promote extracellular functions. The subcellular location of HMGB1 in monocytic cells is known to be dependent on the acetylation status of the nuclear localization signal (NLS) of the HMGB1 protein (Bonaldi et al., 2003). Inflammatory signals promote acetylation of the NLS, leading to cytoplasmic accumulation of HMGB1 in secretory lysosomes in the monocytic cells (Gardella et al., 2002). These secretory lysosomes are subsequently exocytosed when the monocytic cells are triggered by a second inflammatory stimuli. Whether the subcellular location of HMGB1 in Sertoli cells is regulated in a similar way remains to be investigated. Indeed, Sertoli cells share some properties with macrophages including phagocytic activity and production of defensins (Grandjean et al., 1997). The presence of HMGB1 in the interstitial fluid indicates that this protein may be secreted to the interstitial compartment of the testis. However, even though we could not detect intracellular caspase-3 in our interstitial fluid samples, care should be taken when these results are interpreted, because the interstitial fluid was collected by low-speed centrifugation, which may result in some cell damage leading to leakage of intracellular proteins. Although that HMGB1 was also present in the intratubular fluid samples, it cannot be concluded that HMGBI is actively secreted into the luminal compartment, because these same intratubular fluid samples were shown to contain the intracellular protein caspase-3, which indicates leakage of intracellular components during sample preparation. As an alternative to active secretion, HMGB1 may be passively released to the extracellular environment in states of tissue injury and necrosis in vivo. Because HMGB1 has been reported to possess cytokine-like properties (Wang et al., 1999, 2004; Lotze and Tracey, 2005), it can be speculated that the presence of HMGB1 in Sertoli cells serves a dual purpose, that is, to directly kill invading micro-organisms and to act as a co-activator of immune cells. The co-localization of HMGB1 with IL-1 and its potential bi-directional secretion in the adult testis emphasizes the latter function. Moreover, we recently demonstrated that the proinflammatory cytokine IL-18 is expressed in rat germ cells and may be secreted into the intercellular space (Strand et al., 2005). Interestingly, IL-18 and HMGB1 are secreted sequentially by immune surveillance cells following activation (Semino et al., 2005). We have also discovered recently that HMGB1 is expressed in the human adenoid and that the adenoid protein exerts antimicrobial activity against several types of bacteria (Zetterstrom et al., 2002). The adenoid plays the role of a barrier comparable to that of the testis. Thus, in both of these organs, HMGB1 may contribute to the first line of defence against invading bacteria. In conclusion, we have demonstrated here that HMGB1 is expressed by Sertoli cells and germ cells in both the human and the rat testes and that HMGB1 purified from either of these sources possesses antibacterial activity. We propose that this protein constitutes part of an antimicrobial defence system in the testis, designed to protect the developing germ cells from harm by infectious agents.
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We thank Ms Yvonne Löfgren for performing the RT–PCR and Associate Professor Kirsi Jahnukainen and Ms Mirja Nurmio for help with the human material. This project was financially supported by grants from the Swedish Research Council (Proj. No. 2002-5892), European Union (STREP PIONEER FOOD-CT-2005-513991; NoE CASCADE FOOD-CT-2004-506319), Frimurare Barnhuset Foundation in Stockholm, the H. R. H. Crown Princess Lovisa Society for Pediatric Health Care, the Society for Child Care, the Swedish Society of Medicine, the Samariten Foundation and Karolinska Institute.
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References |
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Submitted on February 8, 2006; resubmitted on May 17, 2006; accepted on May 25, 2006.
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