Immunocompetent cells in human testis in health and disease

FERTILITY AND STERILITY

Vol. 48, No.3, September 1987

Printed in U.S.A.

Copyright e 1987 The American Fertility Society

Immunocompetent cells in human testis in health and disease

Mostafa I. EI-Demiry, M.D., Ph.D.*t Timothy B. Hargreave, M.S. * Anthony Busuttil, M.D.:J:

Robert Elton, Ph.D.§ Keith James, D.Sc.* Geoffrey D. Chisholm, Ch.M.*

Western General Hospital and University of Edinburgh, Edinburgh, Scotland

The authors have investigated lymphocyte subpopulations and macrophages in normal human testes and the testes of patients under investigation and treatment for subfertility. Specific monoclonal antibodies were used in an indirect immunoperoxidase technique. In normal tissues, T lymphocytes (Leu 4-positive cells) were present in the rete testis with a preponderance of cells of the suppressor/cytotoxic phenotype. In contrast, no lymphocytes were detected within the peripheral portions of the testis. Cells reacting with the anti-Leu M3 monoclonal antibody, which defines monocytes/macrophages, were detected in appreciable numbers in peripheral testis with a specific location around the seminiferous tubules. HLA-DR-positive cells (human leukocyte antigens-class II [DR] determinants of the major histocompatibility complex) also were identified and showed a similar pattern of distribution to that ofthe Leu-M3 positive cells. While no lymphocytes were seen in the normal peripheral testis, T lymphocytes were detected in testicular biopsies from subfertile patients. Suppressor/cytotoxic T cells (Leu 2a-positive) predominated in patients with oligozoospermia and obstructive azoospermia while T cells of the helper/inducer phenotype predominated in patients with unilateral testicular obstruction and in postvasectomy patients. Sperm antibody measurements correlated with these findings. Fertil Steril 48:470, 1987

Autoimmunity to sperm as a possible cause of infertility in men was initially reported in 1954. 1 To date, most studies in humans on autoimmunity to sperm have involved measuring autoantibodies to sperm antigens. The mechanism of induction of these antibodies is poorly understood and the role of cell-mediated immunity is thought to be minor. The main mechanism of sequestration of sperm within the seminif-

Received February 11, 1987; revised and accepted May 18, 1987. * Department of Surgery/Urology, University of Edinburgh. t Reprint requests: Mostafa El-Demiry, M.B., Department of Surgery, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland. :\: Department of Pathology, Western General Hospital. § Medical Computing and Statistic Unit, University of Edinburgh.

470

EI-Demiry et aI. Immunocompetent cells in human testis

erous tubules is thought to be the blood-testis barrier formed by the tight junctions between Sertoli cells. 2 Immune orchitis can be experimentally induced in animals 3 and in man,4 but is not thought to be a common phenomenon in sub fertile men. 5 Hargreave et al. 6 found no evidence of lymphocytic, macrophage, or plasma cell infiltration in any testicular biopsies from 94 men with sperm density less than 10 million/ml and 9 men with obstructive azoospermia. Furthermore, Bigazzi et al. 7 reported that no human studies have revealed monocytic infiltration to be a characteristic of postvasectomy testicular biopsy specimens. However, Hendry et al. B have reported one patient with high titer of antisperm antibody to have focal round cell infiltrate around the seminiferous tubule. The development of specific monoclonal antibodies to lymphoreticular cell surface markers, as well as highly sensitive immunohistochemical Fertility and Sterility

staining techniques, has made it possible to study immunocompetent cells in tissue sections. Using this approach, we have previously defined the location and distribution of immunocompetent cells within the normal tissues of the male genital tract. 9 In the present study, in an attempt to increase our understanding of the role of cell-mediated immunity in immunologic infertility, we have investigated lymphocyte subpopulations and macrophages in human testis in both healthy and disease states using specific monoclonal antibodies. MATERIALS AND METHODS Normal Testicular Tissues

Specimens of normal human testis were obtained over a 2-year period from eight fertile men with a mean age of 28.7 (range, 19 to 40 years), who had died in road traffic accidents. Relatives had given permission for multiorgan donation for transplantation and research purposes. Testicular Biopsies from Infertile Patients

Testicular biopsies were obtained from 24 patients with a mean age of 33.7 ± 6 years under investigation and/or treatment of male infertility. Ten biopsies were taken from ten men who, after three semen analyses, were found to have a sperm density < 10 X 106/ml. Biopsies also were taken from eight other patients, six of whom were diagnosed as having congenital obstructive azoospermia and two as having unilateral testicular obstruction. An additional six biopsies were taken from six previously fertile, healthy men who had Table 1

undergone vasectomy. Biopsies from patients with testicular obstruction were taken at the time of epididymovasostomy or at the time of vasectomy reversal. In all patients, the presence of agglutinating sperm antibody in serum and in seminal plasma was determined before biopsy using the tray slide agglutination test (TAT). Tissues were snap frozen in liquid nitrogen within 1 to 2 hours of surgical removal and stored at -70°C until used. Representative blocks were prepared from the testis and rete testis from each of the normal specimens. Cryostat sections, 5 JLm in thickness, were cut from each block. Sections were numbered in order, air-dried for 1 to 2 hours, fixed in acetone for 10 minutes at room temperature, and either stained immediately or kept at -20°C until used. Monoclonal Antibodies

A range of mouse monoclonal antibodies,IO-16 with well-defined specificities to human lymphoreticular cell surface markers, was used. These are detailed in Table 1. The optimal dilutions of these antisera and the reliability of the staining procedure were assessed by staining similarly frozen sections of normal human spleen and lymph nodes. These tissues also were used as positive controls in every staining session. Immunoperoxidase Staining

The initial sections in each series were stained with hematoxylin and eosin for histologic assessment. The immunoperoxidase staining was performed using the indirect technique as described previously.9 Briefly, serial sections were incubated

Murine Monoclonal Antibodies Reacting with Human Lymphoreticular Cells

Monoclonal antibody"

Dilutionb

Specificity

Antihuman leukocyte Anti Leu 4 Anti Leu 3a Anti Leu 2a BId Anti Leu M3 -3.9' -24' Anti-HLA-DR

1:50 1:100 1:100 1:100 1:50 1:50 Neat Neat 1:100

All leukocytes All T lymphocytes T-helper/inducer cells T-suppressor/cytotoxic cells B lymphocytes Monocytes/macrophages, Interdigitating, and/or dendritic Cells B cells, monocytes, macrophages, and activated T cells

• Except where indicated, the monoclonal antibodies were obtained from Becton Dickinson Laboratory, Impex Limited, Twickenham, England. b Optimum dilutions vary from one batch to another and should always be determined. Please note that all the antibodies were derived from ascitic fluids except 3.9 and 24, which were Vol. 48, No.3, September 1987

References Beverley, 193010 Ledbetter et aI., 1981 11 Evans et aI., 1981 12 Stashenko et aI., 198013 Dimitriu-Bona et aI., 198314 Hogg et aI., 198615 Lampson and Levy, 198016

tissue culture supernatants. CDr. N. Hogg, Macrophage Laboratory, Imperial Cancer Research Fund, London. d Coulter Electronic Ltd., Luton. The second antibody, the peroxidase conjugate, was used diluted 1:50. El-Demiry et aI.

Immunocompetent cells in human testis

471

with the different primary monoclonal antibodies, followed by peroxidase conjugated rabbit-antimouse antibody. In the case of negative controls, the primary antibodies were omitted. The color reaction was developed by incubating the sections with diaminobenzidine/hydrogen peroxide substrate. Sections finally were counter-stained with hematoxylin, dehydrated, and mounted in DPX (Distrene Plastisier [dibutyl pthalate] Xylene). Immunohistologic Analysis

Sections were examined by light microscopy using 160X magnification. The positively stained cells were identified by their dark brown cell membrane staining. In every case, the number of cells reacting with any individual monoclonal antilymphocyte antibody was determined in ten different fields in identical areas of the serial sections. The results are expressed as the mean ± standard deviation (SD) ofthe number ofthe positively stained cells in a single field (i.e., 0.8 mm 2 ). In the rete epithelium, a differential count of the epithelial cells and the lymphocytes was performed and the prevalence of these lymphocytes has therefore been presented as numbers per 100 epithelial cells. At least 1000 rete epithelial cells were counted, or a minimum of 200 positively stained cells. Wilcoxon rank sum tests were used to compare the mean lymphocyte counts in the groups studied.

RESULTS Normal Testicular Tissues

Hematoxylin and eosin staining of each of the specimens from the eight cadaver donors showed intact germinal epithelium in all the seminiferous tubules with active meiotic activity throughout. Mature spermatozoa were identified in the majority of the tubules. The Leydig cells were normally represented. No inflammatory changes and no vascular abnormalities were seen. The use of the two-layer indirect immunoperoxidase technique provided sufficient intensity of staining to allow the detection of lymphoreticular cells in frozen tissue sections. The reactivity of the primary monoclonal antibodies used in this study with sections of human spleen and lymph nodes (the positive control sections) was confirmed as similar to that previously reported. 17 Cells reacting with the pan antileukocyte (HLe-1) monoclonal antibody were consistently detected in both the peripheral testis and the rete testis. This antibody reacts with all human leukocytes in peripheral blood, spleen, and tonsil, all thymocytes, infiltrating leukocytes in other tissues, B, T, and myeloid celllines. 10 These cells were further characterized using the antilymphocyte and antimonocyte/macrophage monoclonal antibodies. In the normal peripheral testicular tissues, none

Figure 1 Suppressor/cytotoxic T cells (Leu 2a+) in the rete testis. Note that these cells are located mainly inbetween the epithelial cells of the rete testis tubules (cryostat section, immunoperoxidase, X160).

472

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Immunocompetent cells in human testis

Fertility and Sterility

Table 2 Reactivity of Murine Monoclonal Antibodies Specific for T Cells Within the Epithelium of Normal Human Rete Testes (Mean Counts of Intraepithelial LymphocytesllOO Epithelial Cells)

Donor 1 2 3 4 5 6 7 8 All donors

All T lymphocytes (Leu 4-positive) 6.6 5.9 8.6 8.6 6.3 5.4 5.7 8.0

± ± ± ± ± ± ± ±

2.1 1.6 2.2 2.3 1.7 3.2 2.3 3.3

6.9 ± 1.3

T suppressor/cytotoxic cells (Leu 2a-positive) 4.8 4.6 6.8 5.9 3.1 4.6 3.8 6.7

± ± ± ± ± ± ± ±

T helper/inducer cells (Leu 3a-positive) 2.7 ± 1.7 ± 2.2 ± 1.7 ± 1.0 ± 1.1 ± 0.8 ± 1.5 ±

1.2 2.3 1.4 2.0 1.3 1.8 1.9 3.8

5.0 ± 1.3

of the antilymphocyte monoclonal antibodies used gave a detectable reaction, indicating the absence of lymphocytes in these tissues. In marked contrast, T lymphocytes were visualized throughout the rete testes with the anti-Leu 4 monoclonal antibody, which is regarded as a pan T-cell marker. These T lymphocytes were located both within the lining epithelium of the tubules of the rete testes and within the stromal connective tissue of the rete testes (Fig. 1). Studies with subset specific antisera revealed differences in distribution of the T lymphocyte subsets. Thus, T lymphocytes of the suppressor/cytotoxic phenotype (Leu 2a-positive) were more abundant within the lining epithelium (Table 2) while the helper/inducer T cells (Leu 3apositive) predominated in the connective tissue stroma (Table 3). Only a few Bl-positive cells (B cells) were demonstrated, these being located in the connective tissue stroma of the rete testes. Cells staining with the Leu M3 monoclonal antibody, which is believed to bind specifically to monocytes/macrophages,14 were detected in large numbers inbetween the seminiferous tubules. In

1.0 1.1 0.9 1.3 0.7 0.8 0.5 1.0

1.6 ± 0.6

T suppressor/cytotoxic cells to T helper/inducer cells ratios (Leu 2a/Leu 3a) 1.77 2.7 3.09 3.47 3.1 4.18 4.75 4.46 3.44 ± 0.99

order to substantiate these observations, staining also was performed with other monoclonals (designated 3.9 and 24) known to bind to tissue macrophages and cells of interdigitating or dendritic appearance. 15 These antisera also bound cells in the same location. The Leu M3-, 3.9-, and 24-positive cells were largely arranged in a concentric fashion around the tubules and in close proximity to the outer aspect of their walls (the tunica propria) (Fig. 2). No positively stained cells were detected within the component cells of the germinal epithelium. Other positive cells were lying within the interstitial tissues, being abundant around the blood vessels. Positive cells of this type also were seen in the rete testes, within the epithelium, and within the connective tissue stroma. Cells staining with the anti-HLA-DR-monoclonal antibody exhibited a similar location and distribution to that of the Leu M3-, 3.9-, and 24positive cells (Fig. 3). The staining pattern of the anti-HLA-DR antibody overlapped that of the antimacrophage/antisera. This suggests that a majority of these cells express HLA-DR antigens. Unlike

Table 3 Reactivity of Murine Monoclonal Antibodies Specific for T Cells Within the Intertubular Connective Tissues of Normal Human Rete Testes (Average Number of Positive Cells/0.8 mm2 of Intertubular Connective Tissue [X160 ± SD])

Donor 1 2 3 4 5 6 7 8 All donors

All T lymphocytes (Leu 4-positive) 12.3 ± 7.3 ± 14.4 ± 17.7 ± 6.7 ± 6.4 ± 6.8 ± 15.3 ±

2.7 1.7 1.9 2.8 2.8 1.0 1.5 4.7

10.8 ± 4.6

Vol. 48, No.3, September 1987

T suppressor/cytotoxic cells (Leu 2a-positive) 6.3 ± 2.6 ± 5.1 ± 7.5 ± 1.7 ± 2.5 ± 2.2 ± 5.0 ±

2.7 0.5 1.7 3.5 0.8 1.6 1.4 2.9

T helper/inducer cells (Leu 3a-positive) 7.8 ± 6.2 ± 8.2 ± 10.1 ± 4.7 ± 2.9 ± 4.5 ± 12.5 ±

4.1 ± 2.1

2.5 1.5 0.9 1.7 2.4 1.4 1.0 4.8

7.1 ± 3.1

EI-Demiry et al.

T suppressor/cytotoxic cells to T helper/inducer cells ratios (Leu 2a/Leu 3a) 0.8 0.4 0.6 0.7 0.4 0.9 0.5 0.4 0.6 ± 0.2

Immunocompetent cells in human testis

473

Figure 2 Macrophages (Leu Ma + cells) in normal testicular tissue (cryostat' section, immunoperoxidase, X160).

the lining epithelium of the seminiferous tubules (the germ cells and Sertoli cells), which was HLADR-negative, the lining epithelium of the rete testes tubules was HLA-DR-positive. The endothelial surface of the blood vessels also was stained. The anti-T-6 antibody, which reacts with the epidermal Langerhan's cells, gave no detectable reaction with the normal testes. This suggests that

Figure 3 HLA-DR+ cells in normal testicular tissue (cryostat section, immunoperoxidase, X80). 474

EI-Demiry et aI. Immunocompetent cells in human testis

the Leu M3-, 3.9-, and 24-positive cells within the testis are not of the Langerhan's cell type. Testicular Biopsies from Infertile Men

While no lymphocytes were detected in normal peripheral testicular tissues, lymphocytes were detected in all the testicular biopsies from infertile males (24/24). In patients with oligozoospermia, Leu 4-positive cells (T lymphocytes) were detected as single cells in the interstitial tissues between the seminiferous tubules. These T cells were almost always of the suppressor/cytotoxic phenotype (Leu 2a-positive) and were few in number. None of these patients had detectable sperm-antibody in his serum or seminal plasma. Intratesticular lymphocytes were demonstrated in large numbers in patients with testicular obstruction, and they were essentially all Leu 4-positive (T lymphocytes). A few Bl-positive cells (B lymphocytes) were seen. The lymphocytes were found in two locations. Isolated cells were noted between the fibrous tunica propria and the germinal epithelium of the seminiferous tubules. No labeled cells were seen within the germinal epithelium. The other lymphocytes were arranged in small aggregates and follicle-like clusters within the intertubular connective tissue (Fig. 4). In biopsies from patients with obstructive azooFertility and Sterility

Figure 4 (A) Suppressor/cytotoxic T cells (Leu 2a+) in a testicular biopsy from a patient with obstructive azoospermia (cryostat section, immunoperoxidase, X160) (B). Helper/inducer T cells (Leu 3a+) in a testicular biopsy from a postvasectomy patient (cryostat section, immunoperoxidase, X160) (C). Helper/inducer T cells (Leu 3a+) in a testicular biopsy from a patient with unilateral testicular obstruction (cryostat section, immunoperoxidase, X160).

spermia, T lymphocytes of the suppressor/cytotoxic phenotype (Leu 2a-positive) predominated, making the average T suppressor/cytotoxic to the T helper/inducer ratio (Leu 2a+ /Leu 3a+) approximately 4:1 (Table 4). The Leu 2a+ /Leu 3a+ ratios were significantly higher in these patients than in those with unilateral obstruction (P < 0.05) or vas reversal (P < 0.01). However, the total number of Leu 4+ and of Leu 2a+ cells did not differ significantly among the three patient groups. Of these six patients with obstructive azoospermia, only one patient had sperm antibody detected at low titer (1:8) in his serum. In contrast, in biopsies from vas reversal (postVol. 48, No.3, September 1987

vasectomy) patients and patients with unilateral testicular obstruction, the helper/inducer T cells (Leu 3a-positive) predominated. Levels of the Leu 3a-positive cells were significantly higher (P < 0.01) in these patients than in those with obstructive azoospermia (Fig. 5). B lymphocytes (Blpositive cells) also were noted more frequently in comparison to the previous groups. All these patients had sperm antibody detected in high titers in their serum and semen (Tables 5 and 6). DISCUSSION

In this study, mononuclear phagocytes, as defined by the anti-Leu M3, 3.9, and 24 monoclonal EI-Demiry et al.

Immunocompetent cells in human testis

475

Table 4 Reactivity of Monoclonal Antibodies Specific for T Cells with Testicular Biopsies from Patients with Congenital Obstructive Azoospermia (Average Number of Positive Cells per HPF X160 [0.8 mm 3 tissue] ± SD) Sperm antibody TAT titer

All T lymphocytes (Leu 4+ )

T suppressor/cytotoxic cells (Leu 2a+)

T helper/inducer cells (Leu 3a+)

T suppressor/cytotoxic to T helper/inducer cells ratio (Leu 2a+ / Leu 3a+)

12.7 ± 2.4

11.2 ± 2.2

1.8 ± 1.0

6.2

o

o

2

14.9 ± 3.6

11.4 ± 2.2

2.5 ± 1.1

4.6

o

o

3 4 5

15.3 ± 2.9 9.0 ± 1.3 9.7 ± 1.9

8.8 ± 1.5 5.2 ± 1.6 6.6 ± 1.6

2.1 ± 1.0 1.3 ± 0.5 1.7 ± 0.9

4.2 4.0 3.9

o o 8

o o o

6

7.5 ± 1.3

5.1 ± 1.5

2.0 ± 1.1

2.6

o

o

Patient

antibodies were detected in large numbers in normal peripheral testes, but no T or B lymphocytes were demonstrated. In contrast, T lymphocytes

•

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Post vasectomy patients

5

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Patients with obstructive azoospermia

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Figure 5 T lymphocyte subsets in patients with testicular obstruction (mean numbers per HPF X160). Note the predominance of the Leu 3a+ cells in the unilaterally obstructed and the postvasectomy patients.

476

EI-Demiry et al.

Immunocompetent cells in human testis

Serum

Semen

Histologic finding (H & E staining) Normal morphology Chronic inflammatory cell infiltrate Edematous stromaclusters of interstitial cells Normal morphology Edematous stroma Thickening of the tunica propria

were always detected in normal rete testes as well as in testicular biopsies from infertile men. The lack of detectable lymphocytes in normal peripheral testes may be explained by the presence of a physiologic mechanism for the immunologic isolation of spermatozoa: the blood-testis barrier. 2 However, in the rete testes, where the bloodtestis barrier is weak or deficient,2 an alternative mechanism is required to prevent an autoimmune response to sperm differentiation antigens. Such a mechanism could be provided by the relatively high numbers of the suppressor/cytotoxic T cells, observed within the epithelium of the rete testis tubules, which have been implicated in the induction of immunologic tolerance. 18 It should also be noted that T lymphocytes with predominance of the suppressor/cytotoxic phenotype were found to constitute a resident population along the lining epithelium of other genital tract organs, including the vas deferens, the prostate, and the seminal vesicles. 9 The presence in peripheral testes of relatively large numbers of cells reacting with antimonocyte/ macrophage monoclonals was unexpected because such cells have been difficult to identify using conventional histologic techniques. While we cannot entirely exclude the possibility that certain of the monoclonals used cross-reacted with other cells, e.g., fibroblasts, we believe that the present studies indicate that there are significant numbers of macrophages/monocytes within the peripheral testes. These antisera failed to stain cultured human skin fibroblasts or fibroblasts in prostatic tissue and skin. Furthermore, our observations are in agreement with previous histologic studies in man 19 and rat. 20 Also, in the rat testis, large amounts of Interleu kin 1 have recently been detected and this is a macrophage-derived protein. 21 Fertility and Sterility

Table 5 Reactivity of Monoclonal Antibodies Specific for T Cells with Testicular Biopsies from Postvasectomy Patients (Average Number of Positive Cells per HPF 160X [0.8 mm 3 tissue] ± SD) Sperm antibody TAT titer

Patient

All T lymphocytes (Leu 4+)

T suppressor/cytotoxic cells (Leu 2a+)

T helper/inducer cells (Leu 3a+)

T suppressor/cytotoxic to t helper/inducer cells ratio (Leu 2a+/ Leu 3a+)

7 8

13.4 ± 3.0 16.7 ± 2.6

7.8 ± 1.1 8.0 ± 1.3

11.5 ± 1.4 9.7 ± 1.6

0.7 0.8

2048 2048

64 64

9

9.6 ± 1.9

6.0 ± 1.3

6.4 ± 1.2

0.9

1024

32

10

8.9 ± 1.0

6.1 ± 1.6

5.5 ± 1.7

1.1

64

32

11 12

25.2 ± 4.7 20.2 ± 6.6

11.5 ± 2.9 8.4 ± 2.3

16.6 ± 3.3 14.2 ± 3.6

0.7 0.6

256 1024

64 16

The role of these macrophages in testicular tissue is not clear. They may provide an ancillary back-up protection by destroying and removing sperm released following compromise of the microanatomic blood-testis barrier. However, the expression of HLA-DR antigens on these macrophages and the presence of other HLA-DR-positive cells within the testicular tissues indicate their capacity to induce immune responses. I6 The family of antigen-presenting cells include Ia+ (DR-like) macrophages, dendritic cells in lymphoid tissues,22 and Langerhans' cells in the epidermis,23 with the latter two being especially efficient as antigen presenters. Whether these macrophages are functioning as phagocytic or antigen-presenting cells remains to be established. Our results on testicular biopsies from infertile men revealed the presence of lymphocytic infiltration, mainly of the T lymphocytes, in all the patients with testicular obstruction. The suppressor / cytotoxic T lymphocytes predominated in the biopsies from patients with obstructive azoospermia in whom no sperm antibodies were detected in

Serum

Semen

Histologic Finding (H & E staining) Edematous stroma Patchy scarring with tubular atrophy Thickening of the tunica propria Thickening of the tunica propria Edematous stroma Edematous stromamature spermatozoa were not seen

either the serum or semen. Conversely, the helper/ inducer T cells predominated in biopsies from both patients with unilateral testicular obstruction and vas reversal and this may explain, in part, the high titer of antisperm -antibodies noted in the serum and semen of these patients. It is possible that, in the latter patients, the load of antigens was beyond the capacity of the suppressor cells or the low dose tolerance induced after puberty is broken by a supraphysiologic antigen dose after vasoligation. It is now well established that T cells are involved in a complex series of interactions that regulate the immune response. Interactions between the Leu 2a-positive cells and Leu 3a-positive cells are required to induce suppression of a variety of reactions, including immunoglobulin production following antigen stimulation and pokeweed mitogen, or autologous mixed leukocyte-induced reactions.24 Conversely, the Leu 3a subset of cells has been shown to proliferate in the presence of soluble antigens and to induce both B cells to secrete immunoglobulin and precytotoxic cells to become cytotoxic. 25 It also has been demonstrated that, with

Table 6 Reactivity of Monoclonal Antibodies Specific for T Cells with Testicular Biopsies from Patients with Unilateral Testicular Obstruction (Average Number of Positive Cells per HPF 160X [0.8 mm2 tissue] ± SD) Sperm antibody TAT Titer

Patient

All lymphocytes (Leu 4+)

T suppressor/cytotoxic cells (Leu 2a+)

T helper inducer cells (Leu 3a+)

T suppressor/cytotoxic to T helperlinducer cells ratio (Leu 2a + / Leu 3a+)

13

11.4 ± 3.0

5.5 ± 1.6

6.9 ± 1.4

0.8

1042

64

14

26.1 ± 9.4

10.9 ± 2.2

20.1 ± 5.8

0.5

512

32

Vol. 48, No.3, September 1987

EI-Demiry et al.

Serum

Semen

Histologic findings (H & E staining) Intertubular edema. Tendency to sloughing of the germinal epithelium Thickening of the tunica propria

Immunocompetent cells in human testis

477

the appropriate target cells, a subset of Leu 3a+ cells may be cytotoxic as well. Nevertheless, the demonstration that Leu 3a cytotoxic lymphocytes are specific for class II molecules (DR-antigens) indicates a possible mechanism for tissue damage. Such cytotoxic cells could conceivably damage a variety of cells bearing class II antigens, including endothelial cells. Overall, our observation, the infiltration of testicular biopsies from unilaterally obstructed and vasectomized men, increases our understanding of the possible mechanisms involved in antispermantibody production and the pathologic changes observed in the testicular tissue in these patients. The observation that the unilaterally obstructed patients resemble vasectomized patients with regard to the pattern and type of cell infiltrate and the presence of sperm antibodies in their serum and seminal plasma may indicate the clinical significance of unilateral testicular obstruction in subfertile males. In summary, using specific monoclonal antibodies, immunocompetent cells were identified in human testes in health and disease. They were found to have specific patterns of distribution. In normal tissues, T lymphocytes, with a preponderance of the suppressor/cytotoxic phenotype, were demonstrated in the rete testis, while macrophages and HLA-DR-positive cells were located mainly in the peripheral testis and around the seminiferous tubules. In contrast, T lymphocytes were detected in testicular biopsies from subfertile patients. The suppressor/cytotoxic T cells predominated in patients with oligozoospermia and obstructive azoospermia while the helper/inducer T cells predominated in patients with unilateral testicular obstruction and vas reversal. There are a number of ways in which the present work could be extended. First, these studies need to be confirmed in a large series. Of prime importance in this regard are similar investigations in additional patients with detectable sperm antibodies in serum and/or semen. Where possible, further studies with other monoclonal probes would be an advantage, especially those that indicate the activational status of cells of the immune system. Undoubtedly, however, the most pressing need is for studies with a monoclonal antibody that can readily distinguish between the T -suppressor and T -cytotoxic cell subsets. Furthermore, functional studies are also essential but, at present, the availability of tissue, together with technical limitations, render this difficult. 478

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Acknowledgments. We thank Nancy Hogg, Ph.D., Imperial Cancer Research Fund, London, for supplying the 3.9 and 24 antimacrophage antibodies; Graham C. Priestley, Ph.D., for supplying cultured skin fibroblasts; and Mrs. Irene Soutar, for preparing the manuscript.

REFERENCES 1. Wilson L: Sperm agglutinins in human semen and blood. Proc Soc Exp Bioi Med 85:652, 1954 2. Johnson MH: Physiological mechanisms for the immunological isolation of spermatozoa. Adv Reprod Physiol 4:279, 1973 3. Taguchi 0, Nishizuka Y: Experimental autoimmune orchitis after neonatal thymectomy in the mouse. Clin Exp Immunol 46:425, 1981 4. Mancini RE, Amdrada JA, Sarceni D, Bachman AE, Lavieri JC: Immunological and testicular response in man sensitized with human testicular homogenate. J Clin Endocrinol Metab 25:859, 1965 5. Rumke P, Hellinga G: Autoantibodies against spermatozoa in sterile men. Am J Clin Pathol 32:357, 1959 6. Hargreave TB, Busuttil A, Elton RA, Harvey J, Chan A, Chisholm GD: Studies of testicular and epididymal damage in relation to the occurrence of antisperm antibodies. Br J Urol 54:769, 1982 7. Bigazzi PE, Alexander NJ, Silber SJ: Studies on testicular biopsies from vasectomised men. In Vasectomy: Immunological and Pathophysiologic Effects in Animals and Man, Edited by IH Lepow, R Crozier. New York, Academic Press, 1979, p 509 8. Hendry WF, Stedronska J, Hughes L, Cameron KM, Pugh RCB: Steroid treatment of male subfertility caused by antisperm antibodies. Lancet 2:498, 1979 9. El-Demiry MIM, Hargreave TB, Busuttil A, James K, Ritchie AWS, Chisholm GD: Lymphocyte sub-populations in the male genital tract. Br J Urol 57:769, 1985 10. Beverley PCL: Production and use of monoclonal antibodies in transplantation and clinical immunology. In Transplantation and Clinical Immunology, Vol. II, Edited by JL Touraine, J Traeger, H Betuel, J Brochier, JM Dufernard, JP Revillard, R Triau. Amsterdam, Excerpta Medica, 1980, p 87 11. Ledbetter JA, Evans RL, Lipinski M, Cunningham-Rundles C, Good RA, Herzenberg LA: Evolutionary conservation of surface molecules that distinguish T-lymphocyte helper/inducer and T-cytotoxic/suppressor subpopulations in mouse and man. J Exp Med 153:310, 1981 12. Evans RL, Wall DW, Platsoucas CD, Siegal FP, Fikrig SM, Testa CM, Good RA: Thymus-dependent membrane antigens in man: inhibition of cell mediated lymphocytosis by monoclonal antibodies to the TH2 antigen. Proc Natl Acad Sci USA 78:544, 1981 13. Stashenko P, Vadler LM, Hardy R, Schlossman SF: Characterization of a human B lymphocyte specific antigen. J ImmunoI125:1678, 1980 14. Dimitriu-Bona A, Burmester GR, Waters SJ, Winchester RJ: Human mononuclear phagocyte differentiation antigens. I. Patterns of antigenic expression on the surface of human monocytes and macrophages defined by monoclonal antibodies. J ImmunoI130:145, 1983 15. Hogg N, Takacs L, Palmer DG, Selvendran Y, Allen C: The P150, 95 molecule is a marker of human mononuclear Fertility and Sterility

16. 17.

18.

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