1 Departments of Pathology and 2 Urology, The Lady Davis Carmel Medical Center, and the Technion Rappaport Faculty of Medicine, Haifa, 34362 and 3 The National Center of Forensic Medicine, Tel-Aviv, 61490, Israel
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Abstract |
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Key words: cryptorchidism/cytotoxic lymphocytes/epididymis/granzyme B/testis
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Introduction |
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Several studies have characterized the immunophenotype and distribution of immune cells in the testis, as well as in the different regions of the epididymis, of normal and infertile males. In the normal human testis lymphocytes are not usually detected within the seminiferous tubules (El-Demiry et al., 1985). Most intra-epithelial lymphocytes (IELs) of the rete testis, epididymis and vas deferens stain positively for T-lymphocyte markers (Ritchie et al., 1984
). It has been observed that a major proportion of epididymal IELs express the CD8+ suppressor/cytotoxic phenotype (Ritchie et al., 1984
; El-Demiry et al., 1985
). CD8+ lymphocytes include a subset of cells with cytotoxic potential. Recently, antibodies that recognize antigens associated with the cytotoxic phenotype have become available. TIA-1 is a granule-associated, RNA-binding protein which defines a subpopulation of CD8+T-cells with cytotoxic potential (Anderson, 1996
; Medley et al., 1996
). Granzyme B is a serine protease expressed specifically by activated CTLs (Griffiths and Mueller, 1991
). Granzyme B, along with granzyme A and perforin, synergistically triggers apoptosis in target cells by means of granule exocytosis (Hanson and Ley, 1990
). Expression of granzyme B and TIA-1 has been demonstrated by IELs of the normal gastrointestinal tract and the female reproductive tract (King et al., 1993
; Mulder et al., 1997
; Muller et al., 1998
; Oberhuber et al., 1998
; Resnick et al., 1999
). In addition, these cytotoxic markers are expressed by CTLs in several pathological states, including autoimmune and inflammatory diseases, allograft rejection and in several solid tumours and lymphomas (Griffiths and Mueller, 1991
; Oudejans et al., 1996
; Muller et al., 1998
; Yakirevich et al., 1999
). The cytotoxic potential and activation status of IELs in the male reproductive tract has not as yet been assessed.
The aim of this study was to determine whether a subpopulation of testicular or epididymal lymphocytes with cytotoxic potential and activity exists, and to compare the topographic distribution of these cells in normal human testicular tissue with active spermatogenesis with cryptorchid testes with alterations in germ cell development.
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Materials and methods |
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An additional nine cases of postpubertal cryptorchid testes (age range 1639 years) were retrieved from the archives of the Carmel Medical Center between the years 19972000. All of these cases were surgically resected specimens of patients with a history of unilateral cryptorchidism, without prior hormonal therapy, who underwent orchidectomy in order to avoid the risk of cancer. As was shown in our previous study (Yakirevich et al., 1999) the age of archived tissues did not influence the immunoreactivity of the cytotoxic markers. All cryptorchid testes were in the inguinal position. Only those samples that contained the entire testicular specimens with epididymal and vas deferens portions were included in the study. These cases were distributed into two groups according to the histological evaluation of haematoxylin and eosin stained sections. One group included six cases of Sertoli cell-only (SCO). In the second group, the presence of germ cells was confirmed and included nine cases with maturation arrest at the level of the primary spermatocytes (SCA). In three of these cases few spermatids were also detected. Cases with marked tubular sclerosis or atrophy were not included in the study.
Immunohistochemical staining
Immunohistochemical staining was performed according to the following protocol. Consecutive sections from paraffin-embedded tissue blocks were cut at 5 µm, de-paraffinized, and rehydrated with xylene and graded alcohol. Microwave/pressure cooker pretreatment (two cycles at 370 and 160 W, 15 min each) was performed in 10 mmol/l citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min. Sections were blocked with 10% goat serum for 1 h and then incubated with the primary antibody for 1 h at room temperature. The following primary antibodies and dilutions were used: rabbit polyclonal anti-CD3 (1:50 dilution; Dako, Glostrup, Denmark); mouse monoclonal anti-CD8 (clone C8/144B, 1:100 dilution; Dako); anti-TIA-1 (1:100 dilution; Coulter Corp., Hialeah, FL, USA); and anti-granzyme B (clone GrB-7, 1:20 dilution; Monosan, Sanbio, The Netherlands). The labelled streptavidin-biotin-peroxidase method using the Histostain-Plus kit and AEC substrate from Zymed Laboratories (South San Francisco, CA, USA) was employed. Reactive lymph nodes were used as positive controls.
Double immunohistochemistry for TIA-1/CD8 and granzyme B/CD8 was performed on selected specimens. First, mouse anti-TIA-1 (1:100 dilution) or mouse anti-granzyme B (1:20 dilution) monoclonal antibodies were applied for 1 h at room temperature followed by incubation with the EnVision anti-mouse kit (Dako), using 3,3-diaminobenzidine (DAB) as a substrate (brown). Subsequently, the sections were microwave treated (two cycles at 800 W, 5 min each) and blocked with 10% goat serum for 30 min. Mouse anti-CD8 monoclonal antibody (1:100 dilution; Dako) was applied as described above, except for the use of a Histostain-Plus kit with alkaline phosphatase (Zymed) and fuchsin as a chromogen in the second reaction (red).
Assessment of immunostaining and statistical analysis
Cells staining positively for CD3, CD8, TIA-1 and granzyme B were quantified separately into two groupsIELs and those within surrounding stroma. For each group, the number of stained cells was counted per x400 high power field (HPF) using an Olympus BX50 microscope with an F.N.22 system resulting in a 0.55 mm2 field of vision. At least 10 HPFs were randomly selected and counted and a final mean value was given.
Results are expressed as the mean ± SEM. Statistical differences were analysed using the KruskalWallis non-parametric analysis of varience test followed by multiple group comparisons by the adjusted MannWhitney U-test.
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Results |
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Immunohistochemical studies for cytotoxic markers
The results of the immunohistochemical analysis for cytotoxic markers are illustrated in Figures 1 and 2. Lymphocytes were quantified separately within the epithelium (IELs) and within the surrounding stroma. Positively stained cells within blood vessels were not enumerated. The majority of IELs resided within the basal portion of the epithelial layer. Only very rarely were lymphocytes observed within the lumen. TIA-1+ and granzyme B+ cells stained in a distinctly granular cytoplasmic pattern, whereas CD3+ and CD8+ cells exhibited a diffuse membranocytoplasmic pattern (Figure 1
). To determine whether TIA-1+ and granzyme B+ lymphocytes represented CD8+ cytotoxic cells, TIA-1/CD8 and granzyme B/CD8 double immunohistochemical staining was performed (Figure 1D,H
). The vast majority of the cells that exhibited brown granular cytoplasmic staining for TIA-1 or granzyme B also exhibited CD8-positive membranous red staining.
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Rete testis
Increased numbers of CD3-staining lymphocytes were present in the stroma compared with the epithelium in the rete testis of both groups (Figure 2). Although the numbers of CD3+ IELs were higher in normal than in cryptorchid testes, they did not reach statistical significance. Only rare granzyme B+ lymphocytes were detected in normal rete testis (0.5 ± 0.2), whereas lymphocytes within the cryptorchid rete testis did not express granzyme B. The number of stromal CD8+, TIA-1+ and granzyme B+ lymphocytes was significantly greater in the normal as opposed to both cryptorchid groups (Figure 2
).
Epididymis
The number of CD3+ lymphocytes was always greater within the epithelium than within the surrounding stroma at all levels of the epididymis in normal as well as in both cryptorchid groups. The number of intra-epithelial and stromal CD3+ lymphocytes was greater in the cauda than in the more proximal caput and corpus regions of the normal epididymis (94.9 ± 11.6, 42.5 ± 8.2 and 44.1 ± 6.1 for IELs and 73.0 ± 12.0, 37.1 ± 9.6 and 33.3 ± 5.5 for stromal lymphocytes respectively). The distribution of CD3+ cells within the epididymal epithelium of the SCA group resembled the normal group and the number of these cells in the cauda and corpus of the SCA group (66.3 ± 21.9 and 32.6 ± 11.0) was higher than in the SCO group (32.7 ± 8.1 and 19.2 ± 3.0). The number of CD3+ stromal lymphocytes was significantly higher in the normal as opposed to the SCO and SCA groups. The majority of CD3+ lymphocytes were of the CD8+ cytotoxic/suppressor phenotype in the normal, as well as the cryptorchid epididymis, in both the epithelium and stroma. A significant proportion of these cells also expressed the TIA-1+ cytotoxic potential phenotype. Granzyme B immunostaining revealed a subpopulation of IELs and stromal lymphocytes which expressed cytotoxic activity. The number of TIA-1 and granzyme B-positive IELs was significantly greater in the normal compared with the SCO, but not the SCA group in all regions of the epididymis. The number of stromal TIA-1+ lymphocytes was greater in the normal as compared with the SCO and SCA groups. Although the numbers of intra-epithelial and stromal TIA-1+ and granzyme B-positive cells were higher in the SCA than in the SCO group, they did not reach statistical significance. No significant difference was found between the number of stromal granzyme B+ lymphocytes in the caput epididymis, rete testis or seminiferous tubules. However, the total number of intra-epithelial granzyme B+ cells in the normal epididymis increased from the caput to the corpus and cauda (3.5 ± 1.4, 6.1 ± 1.4 and 21.1 ± 4.7 respectively).
The proportion of TIA-1+ and granzyme B+ lymphocytes as compared with the total number of CD8+ lymphocytes were analysed separately within the epithelium and surrounding stroma. The results are presented in Figure 3. The proportion of TIA-1+ cells within the various levels of the epithelium and stroma of the normal and both cryptorchid groups did not show any significant difference. The mean ratio of granzyme B+ to CD8+ IELs was significantly greater in the normal as compared with the SCO cryptorchid epididymis and was greater, although not statistically significant, in the SCA compared with the SCO group. The proportion of granzyme B+/CD8+ cells increased in the distal portions of normal epididymis and reached 0.09 ± 0.04, 0.19 ± 0.07 and 0.37 ± 0.07 within the caput, corpus and cauda of the epididymis respectively. In the SCA cryptorchid group the granzyme B+/CD8+ ratio reached 0.1 ± 0.06, 0.075 ± 0.04 and 0.18 ± 0.1 within the caput, corpus and cauda respectively. In all regions of epididymis the proportion of stromal granzyme B+/CD8+ lymphocytes was higher in the normal than the cryptorchid epididymis; however, no significant difference was found between the different regions of epididymis.
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Discussion |
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Most IELs in the epithelium of the human testicular excurrent ducts express the CD8 suppressor/cytotoxic phenotype (Ritchie et al., 1984; El-Demiry et al., 1985
). Previously, it was observed that CD8+ lymphocytes were the predominant epididymal immune cells in Brown Norway rats (Serre and Robaire, 1999
). In contrast to these and our results, it was demonstrated that a majority of IELs expressed the CD4+ helper phenotype in Lewis and Wistar rats (Hooper et al., 1995
; Flickinger et al., 1997
). This discrepancy may be related to species differences in the main subpopulations of epididymal lymphocytes. In the El-Dimery et al. and Ritchie et al. studies, antibodies were not as yet available for the characterization of lymphocytic cytotoxic potential and activity (Ritchie et al., 1984
; El-Demiry et al., 1985
).
This study demonstrates that in different regions of the male genital tract, a significant proportion of IELs exhibit cytotoxic potential, as determined by TIA-1 immunostaining, whereas a subpopulation of lymphocytes exhibit cytotoxic activity by granzyme B staining. In the normal epididymis, the number of granzyme B+ lymphocytes increased strikingly from the caput region distally to the cauda. Furthermore, as evaluated by granzyme B/CD8 double immunostaining, the proportion of intra-epithelial and stromal granzyme B+ lymphocytes as compared with the total number of CD8+ lymphocytes was significantly higher in the more distal regions of epididymis. The proportion of granzyme B/CD8 was the highest in the proximal vas deferens. Thus, it appears that in addition to a generalized increase in total numbers of IELs in the normal cauda epididymidis as opposed to the more proximal segments, there is a specific increase in a subset of activated granzyme B+ cytotoxic lymphocytes in the cauda epididymidis and proximal vas deferens.
IELs expressing the TIA-1+ and granzyme A or B+ cytotoxic phenotype have been previously demonstrated in the normal oesophagus, stomach, small intestine, colon and endometrium (King et al., 1993; Mulder et al., 1997
; Muller et al., 1998
; Oberhuber et al., 1998
; Resnick et al., 1999
). Similar to our findings, in the above locations the majority of IELs possessed the TIA-1+ cytotoxic potential and only a minor subset were activated granzyme B+ lymphocytes.
The function of IELs is an area of considerable debate. Intestinal IELs are thought to provide a first line of defence against local, mainly viral, antigens (Beagley and Husband, 1998). Cytotoxic IELs have also been implicated in epithelial cell turnover, eliciting apoptosis of intestinal epithelial cells (Beagley and Husband, 1998
). It has been postulated that cytotoxic lymphoid cells within normal endometrium may be involved in epithelial surveillance and in the control of normal placentation (King et al., 1993
). It has also been hypothesized that a resident population of immunocompetent IELs may play some role in segregation of sperm antigens from the general circulation (Dym and Romrell, 1975
). Our observation that the number of cytotoxic TIA-1+ and granzyme B+ lymphocytes is significantly greater in the normal than in the cryptorchid group without spermatogenesis is consistent with this hypothesis. There is considerable absorption of testicular fluid and antigenic products of sperm degradation in different regions of the testicular excurrent ducts. In normal conditions, spermatozoa undergo a process of degeneration in the cauda epididymidis and proximal vas deferens of male mammals (Cooper and Hamilton, 1977
). In our study, the highest levels of the activated granzyme B+ lymphocytes were seen in the cauda epididymidis and proximal vas deferens, i.e. in the region of spermatozoal destruction. It has been proposed (Holstein, 1978
) that spermatophagy (phagocytosis of spermatozoa or fragments of spermatozoa) may occur in the human epididymis under normal conditions in macrophages residing in the lumen. IELs have been observed in direct contact with intra-epithelial macrophages within the normal human and rat epididymis (Wang and Holstein, 1983
; Serre and Robaire, 1999
). In addition, it was shown (Yeung et al., 1994
) that basal epithelial cells of human epididymis expressed a tissue-fixed, macrophage-specific antigen, and ultrastructural similarities to macrophages were observed. Recently, the macrophage-like nature of epididymal basal cells was demonstrated in mice (Seiler et al., 1999
). These observations suggest that sperm-laden macrophages and/or epididymal basal cells may present sperm antigens to lymphocytes.
The `classical' major histocompatibility complex (MHC) class I antigen-presentation pathway is thought to be the major mechanism used by CD8+ CTLs to detect endogenously synthesized foreign proteins, including viral proteins and tumour antigens. Exogenous antigens, including proteins synthesized by extracellular bacteria, fungi and parasites as well as proteins administered during immunization, are processed and presented by class II MHC to CD4+ lymphocytes (Abbas et al., 1997). Recently, an alternative (exogenous) MHC class I pathway was described which may present peptides derived from extracellular antigens to CTLs (Yewdell et al., 1999
). One of the key issues in the presentation of exogenous antigens is the mechanism of `cross-priming', whereby macrophages may recognize and phagocytose dead cells and cell debris and then present exogenous antigens in the context of MHC class I (Bevan, 1976
). The physiological importance of such cross-presentation may be the generation of CTL responses to tumours or virally infected somatic cells as well as inducing tolerance to self antigens (Huang et al., 1994
). We hypothesize that cross presentation of exogenous sperm antigens by macrophages and/or epididymal basal cells in the context of MHC class I occurs in the regions of high spermatozoal destruction (cauda epididymidis and proximal vas deferens).
Lymphocytic activation and proliferation may occur after presentation of sperm antigens to lymphocytes by sperm-laden macrophages. To our knowledge, the activation status of the IELs in the human testicular excurrent ducts has not been well characterized. The study by Wang and Holstein provided morphological ultrastructural evidence that some IELs within the human epididymis exhibited larger nuclei and more abundant cytoplasm with numerous organelles, suggesting an activated status (Wang and Holstein, 1983). Finally, it was demonstrated that membrane-bound granules and other dense bodies are occasionally encountered in the cytoplasm of the IELs in the male reproductive tract of rats and rhesus monkeys (Dym and Romrell, 1975
).
Upon differentiation and activation, CTLs acquire the ability to kill target cells expressing class I MHC molecules. Human testicular germ cells as well as epididymal spermatozoa do not express class I MHC antigens (Anderson et al., 1984). A lack of MHC class I molecules from the spermatozoal surface would suggest that spermatozoa are not a target for CTL-mediated destruction. Some suppressor cells may mediate their effects by killing other cells involved in immune responses. Antigen specific CD8+ lymphocytes can lyse B and CD4+ T-helper lymphocytes, or antigen presenting cells that express foreign protein antigenic determinants in association with their MHC molecules (Abbas et al., 1997
). We suggest that activated CTLs within normal epididymis may control the emergence of sperm-reactive lymphocytes and macrophages, thereby maintaining tolerance to self antigens.
The lymphocytic cytotoxic granule mechanism described in the epididymis in this study may act synergistically with the Fas (APO-1, CD95)/Fas ligand (FasL, CD95L) ligand/receptor system in the testis, which has been implicated in the maintenance of the immune privileged status of this organ (Bellgrau et al., 1995; Francavilla et al., 2000
). In this system, FasL expressed by Sertoli cells in humans and rodents induces elimination of Fas-positive lymphocytes, thereby avoiding recognition of the germ cells by the immune system. Another important function of this system is initiation of the apoptotic death of germ cells which express Fas, thereby regulating the number of germ cells and preventing overload (Lee et al., 1997
). This theory has recently been questioned after the observation that FasL transcription occurs in germ cells, whereas the protein is only displayed on mature spermatozoa, suggesting that the previously described testicular expression of FasL by Sertoli cells is erroneous (D'Alessio et al., 2001
). Thus, the localization and the role of the Fas/FasL system in the testis appears to be controversial.
Although significantly fewer in number than in the normal testis and epididymis, a population of TIA-1+ cells with cytotoxic potential was also detected in the SCO cryptorchid group. However, in this group only very rare granzyme B+ lymphocytes were detected, indicating a lack of CTL activation. It is likely that the lack of germ cells in the SCO cryptorchid testis may contribute to this finding. Activated cytotoxic cells were more numerous in the SCA as compared with the SCO group, although their numbers did not reach levels seen in normal testis. However, the trend observed supports our hypothesis that the presence of spermatozoa or immature germ cells triggers a cytotoxic immune response in the testicular excurrent ducts. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids and residual bodies, contain antigenic components in common with mature spermatozoa (Millette and Bellve, 1977). As these cells are located in the adluminal compartment of the seminiferous epithelium and high levels of immature germ cells may be observed in semen, especially among infertility patients, our findings suggest that immature germ cells or their degradation products may appear along the testicular excurrent ducts and trigger an immune response in the SCA cryptorchid testis.
Previous reports suggested that the morphological and immunological changes in testes seen in experimental animal models of cryptorchidism may be similar to those seen in experimental allergic orchitis (Mengel and Zimmermann, 1982). It was hypothesized that early changes may be due to similar alterations of the bloodtestis barrier, allowing the immunoreactive cells to reach seminiferous tubules and to damage germinal epithelium. However, 3 months after beginning the experiment, no cellular infiltration in the cryptorchid testis was detected (Mengel and Zimmermann, 1982
). To our knowledge, distribution of lymphocytic infiltrates in naturally occurring clinical cryptorchidism has not been addressed. In our study, no immune cells were detected within the epithelium of the seminiferous tubules in cryptorchid testis.
In conclusion, we provide evidence that IELs of the normal human testicular excurrent ducts exhibit markers of cytotoxic potential and activity. Activated cytotoxic lymphocytes may play an important role in the immunological barrier preventing an autoimmune response to spermatozoa.
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Acknowledgements |
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Notes |
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Submitted on April 9, 2001; resubmitted on August 7, 2001
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References |
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accepted on October 8, 2001.