CD 99 and CD 106 (VCAM-1) in human testis

E. Veräjänkorva^1,2, M. Laato ^3,4, P. Pöllänen ^1,5

¹Department of Anatomy, ²Turku Graduate School of Clinical Sciences, ³Department of Medical Molecular Biology and Biochemistry, ⁴Department of Surgery, ⁵Department of Obstetrics and Gynaecology, University of Turku,FIN-20520 Turku, Finland

Asian J Androl 2002 Dec; 4: 243-248           

Keywords: testis; prostate carcinoma; hydrocele; cytokines; adhesion proteins; CD99; CD106; IL-2; IL-6; IL-10; IFN-g; TNF-a

Abstract

Aim: The expression of the cytokines IL-2, IL-6, IL-10, IFN-g and TNF-a and the adhesion proteins CD99 and CD106 was studied in the human testis at the protein level. Methods: The expression of the cytokines and the adhesion proteins was assessed using immunohistochemistry and immunoblotting. Results: None of the cytokines studied was present in the human testis, but CD99 and CD106 (VCAM-1) strongly were expressed in all the testes investigated. CD99 was present in the interstitial tissue of the human testis as well as in the Sertoli cells. The identity of the CD99+ interstitial cells is unclear. CD106 (VCAM-1) was present in Leydig cells as well as the basal parts of the Sertoli cells in the seminiferous tubules. In immunoblotting, CD99 was demonstrated at molecular ratios of 46-57 (kD). This is a novel isoform of the molecule. Conclusion: The human testis produces both CD99 and CD106 and as CD106 mediates cell binding to lymphocytes, it is possible that the human Leydig cells adhere to lymphocytes like the rodent Leydig cells.

Testis is an immunologically privileged site [1-2] but little is known about the local expression of adhesion proteins and cytokines in protein level in the human testis in vivo. In the previous studies it has been shown that the rodent testis produces cytokines and adhesion proteins such as IL-1a-like factor [3], IL-6 [4-5] and IL-11[6] as well as TNF-a [7] and TGF-b[8-10], IFN-a and -g [11], Fas Ligand [12], macrophage migration inhibitory factor (MIF) [13], ICAM-1 (CD54) and VCAM-1 (CD106) in vitro [14] and in vivo [15]. The human testis has been reported to produce IL-1a-like factor in vivo[16] and IL-6 [17] in vitro. In human seminoma, the endothelial cells express VCAM-1 and ELAM-1 and Sertoli cells express ICAM-1 in vivo [18-19]. Sertoli cells of human testes have also been shown to produce CD99[20] and TGF-a [21] in vivo. In addition, the human seminal plasma has been reported to contain large amounts of TGF-b [22]. Also evidence of the production of nerve growth factor (NGF) and NGF receptors [23] as well as insulin-like growth factor-I (IGF-I) and IGF-I receptors[24] in the human testis has been presented.

Several factors, including local suppression of T-lymphocyte function by testicular products [8, 25-26], partial mechanical isolation of autoantigens [27-28], induction of clonal anergy [29] and destruction of T cells by local products like Fas ligand [30-31] have been suggested to prevent the immune responses in the testis. However, only little data exist on how T cells migrate to the testis [32-33] and nothing is known about the role of proteins adhering to lymphocytes in the human testis.

In the present study, the cytokine (IL-2, IL-6, IL-10, IFN-g and TNF-a) and adhesion protein (CD99 and CD106) environment were studied in the human testes to see if the testicular Leydig cells can bind lymphocytes to their surface like in the rodents and if the cytokine balance in the human testis prefers cellular or humoral immunity.

2 Materials and methods

Three testes from different individuals were obtained from patients suffering from either prostate carcinoma (n=2) or hydrocele (n=1) and therefore undergoing orchiectomy. Neither of the patients had received any anti-gonadotropin medication prior to the castrations. The patients were treated at the Turku University Central Hospital. Permissions for tissue donation and to use organs for research purpose were granted by the Hospitals and University's Joint Ethical Board (August, 2000). The testes were freezed in liquid nitrogen and stored in -70 for later use.

Monoclonal antibodies against mouse cytokines and adhesion molecules were used as primary antibodies. The antibodies were as follows: rat-anti-human IL-2 (IgG_2a; Pharmingen, clone MQ1-17H12), rat-anti-human IL-6 (IgG_2a; Pharmingen, clone MQ2-6A3), rat-anti-human IL-10 (IgG_2a; Pharmingen, clone JES3-19F1), mouse-anti-human IFN-g (IgG₁; Pharmingen, clone B27), mouse-anti-human TNF-a (IgG₁; Pharmingen, clone Mab11), mouse-anti-human CD99 (IgG_2a, k; Pharmingen, clone TÜ2) and mouse-anti-human CD106 (IgG₁, k; Pharmingen, clone 51-10C9). Monoclonal antibodies for the hapten trinitrophenol (TNP) were used as nonrelevant, negative control antibodies. The antibodies were: mouse-anti-TNP (IgG₁; Pharmingen, clone 107.3) and mouse-anti-TNP (IgG_2a, k; Pharmingen, clone G155-178). The antibodies bind in the used conditions specifically to their epitopes in the following tissues: IL-2 and IL-10: human tonsils [34]; IL6: human monocytes [35]; TNF-a: human monocytes [36]; CD99: human thymocytes and T-cells [37]; CD106: human endothelial cells [38].

2.3 Immunohistochemitry with avidin-biotin-peroxidace method

Frozen sections, 2-5 mm in thickness, were cut in a cryostat. They were air-dried briefly at room temperature and then fixed in cold acetone (-20 ) for 1~2 minutes. After airdrying the sections were stored at -20 . Just before use, sections were soaked in TBS for 310 minutes. Then the sections were dehydrated in a decreasing methanol series (70 %, 96 % and 100 %). After that the sections were incubated in 0.3 % H₂O₂ in methanol for 30 minutes. Then the sections were rehydrated in an increasing methanol series (100 %, 96 % and 70 %). After that the sections were washed in TBS for 35 minutes. Then the non-specific binding sites were blocked by incubating the sections either in 2 % normal horse or rabbit serum in TBS for 30 minutes. Then they were incubated with the primary antibodies (diluted into the concentration of 0.5 mg/mL in TBS) for 60 minutes. Then the sections were washed in TBS for 35 minutes and incubated for 60 minutes with the secondary antibody with either peroxidace-conjucated horse-anti-mouse IgG (Vector Laboratories, Inc., CA, USA; pk 4004) or  peroxidace-conjucated rabbit-anti-rat IgG (Vector, pk 4002) for 60 minutes. One drop of antibody was diluted in 10 mL of 1 % BSA (Sigma, St. Louis, MO, USA) in TBS. Then the sections were washed in TBS for 35minutes. After that, the sections were incubated for 30 minutes in the VECTASTAIN ABC Reagent^® , diluted in TBS. Sections were washed again with TBS for 35 minutes and then incubated in 3,3'-diaminoben-zidine (0.5 mg/mL; Sigma, cat# D-5905) containing 1,3-diaza-2,4-cyclopentadiene (0.77 mg/mL; Sigma, cat# I-0125) and 0.04 % H₂O₂ in TBS for five minutes. Then the sections were washed briefly in H₂O and stained in Mayers hematoxylin for 30 seconds. Finally, the sections were incubated in xylen for 10 minutes. Sections were mounted in Mountex^® (Histolab Products Ltd, Göeborg, Sweden, cat# 00851) and stored in room temperature. The sections were examined and photographed under microscope.

2.4 Antigens for western blotting

Testicular tissue samples (n=3) were prepared for immunoblotting as modified from an original work [39]. Tissue homogenates were prepared in ice-cold suspension buffer (0.1 mol/L NaCl; 0.01 mol/L Tris, pH 7.6; 0.001 mol/L EDTA, pH 8.0) supplemented with 0.3 mg/mL phenylmethylsulfonyl fluoride (Sigma, cat# P-7626) and soybean trypsin inhibitor (Sigma, cat# T-9003) to avoid proteolysis. Briefly, 1 g of tissue was placed in 3 mL of suspension buffer and homogenised. After centrifugation at 250g for 15 minutes, the supernatant was collected and centrifuged again at 10 000 g for 30 minutes. The salts were removed from the supernatant in a Sephadex G-25^®  column (PD-10, Pharmacia Biotech, Uppsala, Sweden). The eluate was freeze-dried. The protein was diluted to approximately 10 mg/mL in 2 Laemmli solution (1 % SDS, 10 % glycerol, 0.01 % bromophenol blue and 2 % 2-mercapto-aethanol [Fluka AG, Switzer-land, cat# 63690] in 50 mmol/L Tris buffer, pH 6.8). The samples were boiled for 5 minutes.

2.5 Immunoblotting

Denatured 12 % SDS-polyacrylamide mini-gels were prepared and 30~40 mL samples were loaded into the wells. Low-molecular weight markers (Pharmacia, cat# 17-0446-01) were run parallel to the samples. Gels were run with a 150 mA current and after electrophoresis proteins were transferred to the nitro-cellulose filter for 60 minutes using a 400 mA current. The nitro-cellulose filter was stained with Ponceau S and each separate line was cut off. Strips were blocked with saline containing 2 % bovine serum albumin (BSA) and 0.2 % sodium azide (Merck, Darmstad, Germany, cat# 6688) and then incubated at 4 with immunosera diluted to 1:50 with PBS containing 2 % BSA and 0.2 % Triton X-100 (Acros Organics, NJ, USA, cat# 21568-0010) for 60 minutes. After incubations the strips were washed three times with PBS and then incubated for 60 minutes in 1:500 dilution with either horseradish peroxidase-conjugated rabbit-anti-rat IgG (20 mg/mL; Dakopatts, Glostrup, Denmark, cat# P162) or horseradish peroxidase-conjugated rabbit-anti-mouse IgG (2.6 mg/mL; Dakopatts, cat# P0260) in PBS containing 2 % BSA and 0.2 % Triton X-100. Strips were washed again with PBS and then allowed to react with 0.6 mg/mL 1,3-diaza-2,4-cyclopentadiene and 0.03 % H₂O₂ in 0.05 mol/L Tris (pH 7.6) for 10 minutes. Reactions were stopped with PBS containing 0.2 % sodium azide and the strips were blotted dry before photographing.

3 Results

3.1 Immunohistochemistry of cytokines and adhesion proteins in human testis

None of the cytokines studied (IL-2, IL-6, IL-10, IFN-g and TNF-a) were expressed in the human testis, where as CD99 and CD106 (VCAM-1) were present in all three testes investigated. CD99 (Figure 1) was produced in the basal compartment of the seminiferous tubules and weakly in some parts of the intratestical tissue. In addition, it appeared that CD99 was expressed only in some sectors in the seminiferous tubules of the human testis (Figure 1b) suggesting a stage-specificity. CD106 was present in the interstitial tissue of the human testis and in some parts of the basal compartment of the somniferous tubules (Figure 2a,c), the endothelium of testicular blood vessels as well as the adjacent tissue of the vessels (Figure 2b, d).

Figure 1. A) CD99 in basal compartment of seminiferous tubules (k) as well as in interstitial tissue (t), immunohistochemistry on frozen section ( 900); B) localisation of CD99 in seminiferous tubules, note the positive (k) and the negative (u) sectors, immunohistochemistry on frozen section (  1200); C) CD99+ cells in basal compartment of somniferous tubules (k), immunohistochemistry on frozen section ( 900); D) CD99+ cells in seminiferous tubules (k) as well as in interstitial tissue ((u), immunohistochemistry on frozen section, (  1800); E) TNP (IgG2a) staining of human testis, immunohistochemistry on frozen section ( 900); F) TNP (IgG2a) staining of human testis, immunohistochemistry on frozen section (  1800).

Figure 2. A) CD106 in interstitial tissue (*) and in seminiferous tubules (t), immunohistochemistry on frozen section (  900); B) CD106 in endothelium (k) and adjacent tissue (u) of testicular vessels, immunohistochemistry on frozen section (x 900); C) CD106 in interstitial tissue (*) and in seminiferous tubules (t), immunohistochemistry on frozen section (  1800), D) CD106 in endothelium (k) of a testicular vessel, immunohistochemistry on frozen section ( 1800); E) TNP (IgG1) staining of human testis, immunohistochemistry on frozen section ( 1800); F) TNP (IgG1) staining of human testis, immunohistochemistry on frozen section ( 900).

All cytokines studied (IL-2, IL-6, IL-10, IFN-g and TNF-a) as well as CD106 proved negative in immuno-blotting. CD 99 was present in all the three testes investigated in immunoblotting. The molecular ratios were 46kD~57kD.

Figure 3. Immunoblots of three human testes; CD99, TNP (IgG_2a), CD106, TNP (IgG₁) used as primary antibodies. Only PBS was used on the fifth strip.

4 Discussion

These results suggest that CD99 and CD106 (VCAM-1) are constitutionally expressed in the human testis. In previous reports, CD99 has been reported to exit as two molecular bands, 30 and 32 kD in Mr, in immunoblotting[40]. In the present study, a new 46 kD~57 kD, novel isoform of CD99 molecule was found in the human testis. CD99 is a cell surface glycoprotein and it is believed to be involved in cell adhesion [41], but quite little is actually known about its role and function. In this study, it was shown that CD99 is produced in some parts of the interstial tissue of the human testis as well as in the basal parts of the seminiferous tubules, suggesting that interstitial cells may bind to human T cells migrating to the human testis and thus be affected [42]. If so, they might be more prone to secrete T_H1-type cytokines [42] because of the CD99 interaction, if none of the other lymphocyte regulating substances of the testis prevents this[8, 43]. The identity of the CD99+ interstitial cells is unclear, but the CD99+ cells in the seminiferous tubules could be Sertoli cells according to their morphology and the previous studies [20]. However, it is not excluded that some of the CD99+ cells might be germ cells as well, because in some cases the staining of CD99 on the seminiferous tubule cross-sections is sector-like (Figure 1b). This would suggest stage-specificity, as the spermatogenic cells progresses like a spiral along the wall of the seminiferous tubules in the humans.

The present results show that CD106 (VCAM-1) was present in the interstitial tissue of the human testis as well as in the basal parts of the seminiferous tubules. CD106 has been previously reported to be produced by the Leydig cells in the mouse testis [15]. The human and the mouse testes seem to be similar in this respect. The fact that CD106 was found in immunohistochemistry but not in immunoblotting could be due to a smaller amount of the CD106 present in the testis than for example CD99 or instability of the human CD106 in the reducing conditions of electrophoresis.

The rodent Leydig cells are able to bind both normal and malignant lymphoblasts on their surface [44-46] and there is evidence that the Leydig cells can suppress lymphocyte proliferation in co-cultures [47]. In fact, it has been proved that the mouse Leydig cells bind to lymphocytes through VCAM-1 (CD106) protein [15]. The present results suggests that also the human Leydig cells may bind lymphocytes infiltrating into the testis through CD106-CD49d/CD29 interaction. Furthermore, on the basis of the literature, it is probable that the human Leydig cells can also regulate T lymphocyte function through the CD106-CD49d/CD29 interaction. This may be of significance, as in children's acute lymphoblastic leukaemia, the disease relapses third often in the testis, after the bone marrow and brain [48]. Still further, the Leydig cell-lymphocyte interaction may be of significance also for prevention of immune responses against the autoantigenic germ cells.

In conclusion, the CD99 and CD106 (VCAM-1), two adhesion proteins able to affect lymphocyte function are expressed in the human testicular interstitial tissue. The role of the interaction of these adhesion proteins with their lymphocyte counterparts remains to be studied in more detail in the future. In addition, CD99 is expressed in the human testis as a novel isoform.

Acknowledgements

This study was supported by the Turku Duodecim Foundation, Eemil Aaltonen Foundation, Oskar öflund Foundation, Jalmari and Rauha Ahokas Foundation, Paulo Foundation, Emil and Blida Maunulas as well as Aili Salos funds of the Medical Faculty of the University of Turku, the Turku Graduate School of Clinical Sciences.

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Correspondence to: Dr. Esko Veräjänkorva, University of Turku, Institute of Biomedicine, Department of Anatomy, Kiinamyllyn-katu 10, FIN-20520 Turku, Finland.
Tel: +358-2-333 7364, Fax: +358-2-333 7352
E-mail: esolve@utu.fi
Received 2002-09-10 Accepted 2002-11-22