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a Departments of Pharmacology and Therapeutics and of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada H3G 1Y6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Brown Norway rat is an excellent model to study aging because of its long life span; when no other systemic disease is apparent, remarkable changes occur in the testis [17, 18]. The histology of the epididymal epithelium of the Brown Norway rat is also severely affected as animals age [19]. Intriguingly, during aging there is a dramatic increase in the number of halo cells, as well as numerous trails of immune cells, apparently leaving the blood circulation to reach the tissue epithelium [19]. These observations suggest a possible age-related activation of the immunocompetent cells in the epididymis.
The major cells involved in immune responses are the monocytes, macrophages, and lymphocytes (T and B) [20]. The T lymphocytes can be divided in two subtypes: the cytotoxic T cells and the helper T cells. CD8 is a marker of cytotoxic T cells and CD4 is a marker of helper T cells [21, 22]. Both lymphocytes and macrophages are present in the mammalian male reproductive tract (human [10, 16,23], rat [14, 15, 24], mouse [25]). The immunocompetent cells present in the epididymis have received very little attention. Consequently, the current literature is sparse, and offers divergent, at times contradictory, reports on the predominant cell types present and their distribution in the epididymis.
We hypothesized that halo cells consist of the main types of immune cells and that their distribution in the epididymis changes during aging. To verify this hypothesis, markers for lymphocytes and monocytes-macrophages were used. Our first goal was to clarify the nature and longitudinal distribution of the halo cells in the epididymal epithelium. Our second goal was to assess the effects of age and luminal content on the distribution of these immunocompetent cells in the epithelium and interstitial tissue of each segment of the epididymis of Brown Norway rats.
The present study demonstrates that halo cells consist of helper T lymphocytes, cytotoxic T lymphocytes, and monocytes. Furthermore, we found segment-specific increases in the number of each type of immune cell assessed with increasing age. In addition, in aged rats, we found a dramatic effect of luminal content on the recruitment of cytotoxic T lymphocytes and monocytes-macrophages into the epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Brown Norway rats aged 3, 12, 18, and 24 mo were purchased from the National Institute on Aging (Bethesda, MD) and supplied by Harlan Sprague Dawley Inc. (Indianapolis, IN). Rats were housed at the McIntyre Animal Resources Center, McGill University, under controlled light (14L:10D) and temperature (22°C); animals had free access to food and water.
Tissue Preparation for Light Microscope Immunocytochemistry
Rats were anesthetized with an i.p. injection of sodium pentobarbital (Somnotol; Steris Laboratories Inc., Phoenix, AZ). The epididymides were fixed with Bouin's solution via perfusion through the abdominal aorta. Retrograde perfusions were done to obtain optimal fixation of the initial segment and caput epididymidis, and prograde perfusions were used to fix the corpus and cauda epididymidis. For the retrograde perfusion, first the common iliac artery on the left side and then the one on the right side were clamped, near the junction of the abdominal aorta; a 18-gauge, 1.5-inch needle was inserted face up at the point of the junction and pointed in the cranial direction, and perfusion with saline was begun. The aorta and inferior vena cava, cranial to the kidney and below the diaphragm, were clamped immediately. The left renal vein, which should have become inflated, was opened in order to allow efflux of saline. When the effluent was free of blood, the perfusion solution was switched to Bouin's fixative for 10 min. For anterograde perfusions, the common iliac arteries were not clamped, and the needle was inserted in the abdominal aorta pointing in the caudal direction; otherwise, the same procedure was followed. After perfusion, epididymides were left for 48 h in Bouin's fixative, dehydrated, and embedded in paraffin. Serial longitudinal sections of 5 µm were cut on a microtome and mounted on glass slides.
Immunostaining
Sections were deparaffinized with xylene and rehydrated in graded alcohol solution. Endogenous peroxidase activity was neutralized using 70% alcohol containing 1% (v:v) hydrogen peroxide. Residual picric acid was inactivated using 70% alcohol containing 1% lithium carbonate. After hydration, free aldehydes were blocked by bathing the sections for 5 min in 300 nM glycine. Immunohistochemical staining of the sections was done using a Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). Primary antibodies used for the immunocytochemical staining of cells present in the epididymal epithelium and interstitial tissue were as follows: anti-CD4 to label helper T lymphocytes, anti-CD8 to label cytotoxic T lymphocytes, anti-ED1 to label monocytes and macrophages, anti-RLN 9D3 to label B lymphocytes, and antiGST Yf to label basal cells. Goat anti-human CD4 (c-18; Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used at a dilution of 1/50; this polyclonal antibody was raised against an epitope corresponding to an amino acid sequence mapping at the carboxyl terminus of the CD4 precursor of human origin, which differs from that of the rat sequences by a single amino acid and cross-reacts with the rat antigen (Santa Cruz technical data sheet). Goat polyclonal anti-rat CD8-
(Santa Cruz Biotechnology), raised against an epitope corresponding to an amino acid sequence mapping at the carboxyl terminus of the rat CD8 alpha chain precursor, was used at a dilution of 1/100. Mouse anti-rat ED1 (Serotec USA, Washington, DC) was used at a dilution 1/100; this antibody recognizes a cytoplasmic antigen in monocytes and most macrophages. Mouse anti-rat RLN-9D3 (Seikagaku America Inc., Rockville, MD) was used at a dilution 1/50, and rabbit anti-rat GST Yf (kindly provided by Dr. J. Hayes; University of Edinburgh, Scotland) was used at a dilution of 1/100. Briefly, the sections were blocked by incubation with diluted normal serum for 30 min at room temperature. Sections were incubated for 18 h at 4°C with primary antiserum diluted in PBS. Sections were then incubated for 30 min with appropriate (anti-goat, mouse, and rabbit) diluted biotinylated secondary antibody solution and incubated for 30 min with Vectastain Elite ABC reagent. Finally, sections were incubated in diaminobenzidine (DAB) solution (Vector Laboratories) until stain developed, rinsed in water, and counterstained with eosin or methylene blue.
Cell Counting
Stained cells were studied in five regions of the epididymis: initial segment, caput, corpus, proximal cauda, and distal cauda; distinctions were based on morphological characteristics [19]. Epididymal sections from 5 to 6 rats per age group were examined by light microscopy at x400 with a green interference filter to enhance contrast (Laborlux D; Leitz Wetzlar, Montreal, Canada). A grid (2.6 mm2) was placed in the center of three cross sections of the epididymal tubule in each segment of the epididymis. In the distal cauda epididymidis, the area covered by the grid was not large enough to include three cross sections of tubules; therefore, in this region the grid was placed in the cross sections of two tubules. Immunomarked cells located within the area covered by the grid were counted in both the epithelium and the interstitium [Ab+]. In addition, we counted halo cells that did not stain [Ab-halo] and the nonstaining cells [Ab-] located in the interstitium region covered by the grid. The grid was moved randomly, and this procedure was repeated 10 times per segment for each animal. Thus the number of cells counted per segment (cell concentration) represents the number of cells in an area equivalent to 0.065 mm2 (10 fields of a grid that is 2.6 mm2 viewed at a x400 magnification).
Size of ED1-Positive Cells
The effect of age on the size of ED1-positive cells was determined in 5–6 animals per age group using light microscopy (x400). A calibrated linear scale was placed in the x10 eyepiece of the light microscope (Laborlux D; Leitz Wetzlar). The size of monocytes-macrophages are reported to range from 15 to 80 µm [26, 27]. We classified ED1-positive cells as either "small" (less than 20 µm; possibly monocytes), "medium" (20–40 µm, most likely activated macrophages), or "large" (more than 40 µm, probably mature macrophages) [27]. We classified 100 ED1-positive cells in the epithelium and in the interstitial tissue in each epididymal segment for each animal.
Statistical Analysis
To assess the nature of halo cells, statistical evaluation was done using Student's t-test (null hypothesis Ho = 1). One-way ANOVA followed by Tukey's test was employed to detect significant age effects and luminal content effects on the distribution of CD4-, CD8-, and ED1-positive cells. The level of significance was taken as p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies directed against monocytes-macrophages and lymphocytes, used on sections of the epididymis of Brown Norway rats, revealed the presence of these cells in both the epithelium and interstitial tissue of the epididymis at all ages (Figs. 1–3). (Changes with age in the number of these cells in different epididymal segments are shown in Figure 6.)
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ED1-Positive cells were found throughout the epididymal epithelium and interstitium at all ages and were frequently located in the lower half of the epididymal epithelium close to the basement membrane (Fig. 1). No ED1-positive cells were seen in the lumen at any age. A segment-specific effect of age and luminal content was noted for ED1-positive cells. Results for the initial segment and caput and corpus epididymidis were similar but differed from those for the cauda epididymidis; therefore, only representative sections from the corpus (Fig. 1, A–C) and distal cauda (Fig. 1, D–F) epididymidis are shown. In the initial segment, caput, and corpus, the number of ED1-positive cells was increased between 3 mo (Fig. 1A) and 18 mo (Fig. 1, B and C). In addition, in these segments, there was a strong immunolabeling of large cells in the epithelium of aged rats that had very few spermatozoa in the lumen (Fig. 1C). In contrast, neither age nor luminal content appeared to affect the number or the size of ED1-positive cells in the distal cauda epididymidis (Fig. 1, D–F). It is of interest to note that ED1-positive cells were often in direct contact with nonstaining halo cells in the aged Brown Norway rat.
CD4-positive cells were small round cells and were found throughout the epithelium at all ages (Fig. 2; corpus, A–C, distal cauda epididymidis, D–F, as representative sections). These cells were located between epithelial cells, usually close to the basement membrane, but were occasionally found up to the apical point of tight junctions between principal cells. They were never seen in the lumen. A segment-specific effect of age was seen in the distribution of CD4-positive cells. There was no change in the relative number of CD4-positive cells in the initial segment and caput, corpus, and proximal cauda epididymidis between 3 and 12 mo (Fig. 2, corpus, A and B). In contrast, an increase in the number of CD4-positive cells was apparent as early as 12 mo in the epithelium of the distal cauda epididymidis (Fig. 2, D and E). However, by 24 mo the number of CD4-positive cells was increased along the entire epididymis (Fig. 2; corpus, C, and distal cauda, F) except for the caput region.
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As was the case for helper T lymphocytes (CD4-positive cells), cytotoxic T lymphocytes (CD8-positive cells) were small round cells, found throughout the epithelium, at all ages (Fig. 3; caput, A–C, and distal cauda epididymidis, D–F, as representative sections). These cells were located at various heights between epithelial cells. They also appeared to be stopped at the tight junction level and were never seen in the lumen. A segment-specific effect of age and luminal content was found in the distribution of CD8-positive cells. The number of CD8-positive cells was increased in the distal cauda epididymidis as early as 12 mo (Fig. 3, D and E), whereas it was unchanged in the remaining segments (Fig. 3, A and B; caput). By 24 mo, the incidence of CD8-positive cells was higher along the entire epididymis (Fig. 3; caput, C, and distal cauda, F).
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In contrast, B lymphocytes were very rarely seen in the epididymal epithelium of 3-mo-old rats and represented less than 1% of immune cells. B lymphocytes were occasionally present at older ages and constituted approximately 5% of immune cells (Fig. 4; initial segment, A, and proximal cauda, B, as representative sections).
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As for T lymphocytes, B lymphocytes were small round cells, often located near the base of the epithelium. They were never seen in the lumen.
Immunostaining of Epididymal Basal and Halo Cells
Basal cells Previous studies by Yeung and coworkers (human [10]) have shown intriguing similarities between basal cells and macrophages. To assess whether basal cells are immune cells in the Brown Norway rat, an antibody directed against the Yf subunit of the glutathione S-transferases (GST), which was previously shown to stain basal cells specifically in the corpus and cauda epididymidis (rat [8]), and antibodies against monocytes-macrophages and lymphocytes were used. The ED1 antibody distinguishes an intracytoplasmic antigen in monocytes, tissue macrophages, and free macrophages (rat [28, 29]). Basal cells from the corpus epididymidis stained with GST Yf, at all ages, as shown by the intense immunolabeling of flat elongated cells located at the base of the epithelium (Fig. 5, corpus, A and B; 3 and 18 mo, respectively). In the epithelium of young rats, occasional small cells were stained with the antibody against monocytes-macrophages; however, the number and localization of these cells did not coincide with that of basal cells (Fig. 5C). In aged rats, more ED1-positive cells were present in the epithelium; however, basal cells did not react with ED1 antibody (Fig. 5D). In addition, basal cells did not stain with antibodies against T lymphocytes or B lymphocytes (data not shown); this is consistent with the size and epithelial localization of these cells shown in Figures 2, 3, and 4.
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Halo cells We hypothesized that helper T lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), and monocytes-macrophages (ED1+) comprise the halo cell population. According to our hypothesis, [CD4+] + [CD8+] + [ED1+] = [total halo]. The total number of halo cells present in the epididymal epithelium can therefore be calculated with this formula: [Ab+] + [Ab-halo]= [total halo]. For each animal (n = 4 per age group), we counted [CD4+] and [CD4-halo], [CD8+] and [CD8-halo], and [ED1+] and [ED1-halo].
If our hypothesis is correct, then we should expect, for any epididymal segment:
We found that the average number of halo cells was equivalent to the sum of ED1-, CD4-, and CD8-positive cells in the epididymis of 3- and 12-mo Brown Norway rats (Ho = 1; 3 mo, 1.04 ± 0.11; 12 mo, 1.08 ± 0.07). A small but significant difference was found at older ages (Ho = 1; 18 mo = 1.26 ± 0.04; 24 mo = 1.15 ± 0.11). However, ED1-positive cells included small, medium, and large cells. These large cells do not fit the description of halo cells, i.e., small round cells with a pale rim of cytoplasm. Thus, when we excluded large halo cells from the equation, the combined number of ED1-, CD4-, and CD8-positive cells was then equivalent to the number of halo cells for age 3, 12, and 24 mo (Ho = 1; 3 mo, 1.04 ± 0.11; 12 mo, 1.07 ± 0.07; 24 mo = 1.05 ± 0.12). A borderline significance was found at 18 mo (Ho = 1; 18 mo, 1.20 ± 0.05; p = 0.03).
Effects of Age on the Number of Immune Cells in the Epithelium
There was a segment-specific increase with age in the number of monocytes-macrophages (ED1-positive cells), helper T lymphocytes (CD4-positive cells), and cytotoxic T lymphocytes (CD8-positive cells) present in the epididymal epithelium (Fig. 6, A–C). The incidence of monocyte-macrophages was fairly constant along the epididymal epithelium of 3-mo-old Brown Norway rats (Fig. 6A). In the initial segment and caput and corpus epididymidis, there was no significant change in the number of monocytes-macrophages between 3 and 12 mo of age. However, there was a major increase by 18 mo in these segments; this increase was sustained at 24 mo. In addition, by 24 mo, the number of monocytes-macrophages became significantly elevated in the proximal cauda epididymidis, whereas the number of monocytes-macrophages was not affected by age in the distal cauda epididymidis (Fig. 6A).
The number of helper T cells (CD4+) slowly increased between 3 and 18 mo and became significantly higher by 24 mo in the initial segment (Fig. 6B). Although there was a trend toward an increase in the number of helper T lymphocytes with age in the caput epididymidis, no significant effect of age was noted. In the corpus and proximal cauda epididymidis, there was a significant increase in the number of helper T cells at 18 and 24 mo, while in the distal cauda epididymidis, this significant increase was seen as early as 12 mo and was sustained at 18 and 24 mo (Fig. 6B).
The number of cytotoxic T cells (CD8+) was unchanged between 3 and 12 mo but was significantly increased by 18 mo in the initial segment (Fig. 6C). In the caput and corpus epididymidis, the concentration of cytotoxic T cells increased slightly, but not significantly, between 12 and 18 mo. However, a dramatic increase of greater than 400% relative to 3 mo was found at 24 mo. In the proximal cauda epididymidis, the number of cytotoxic T cells was increased significantly at 18 and 24 mo as compared with 3 mo. As for helper T lymphocytes, there was a significant increase of cytotoxic T lymphocytes in the distal cauda epididymidis at 12 mo; this increase was sustained at 18 and 24 mo.
Effect of Age on the Number of Immune Cells in the Interstitial Tissue
To develop further insight into the source of immune cells in the epididymal epithelium, the number of immune cells in the interstitial region surrounding the epithelium was assessed. There was no significant effect of age on the number of monocytes-macrophages in the interstitial tissue located in the immediate proximity of the epididymal tubule at all ages. It is noteworthy that there was a trend to a lower concentration at 12 mo in the initial segment and caput, corpus, and proximal cauda epididymidis as compared with 3 mo, followed by a return at 18 and 24 mo to values comparable to those found at 3 mo of age (corpus as representative section: 3 mo, 30 ± 5; 12 mo, 19 ± 3; 24 mo, 27 ± 8). In contrast, there was a steady number of monocytes-macrophages in the distal cauda epididymidis (3 mo, 19 ± 5; 24 mo, 17 ± 4). The number of helper T lymphocytes present in the interstitial tissue was not affected by age in any epididymal segment (caput 3 mo, 25 ± 1, 24 mo, 35 ± 12; corpus 3 mo, 21 ± 5; 24 mo, 30 ± 6, as representative sections); except for the distal cauda epididymidis (distal cauda 3 mo, 9 ± 1; 24 mo, 20 ± 4, p < 0.02). The number of cytotoxic T lymphocytes present in the intertubular space was not significantly affected by age in any epididymal segment (caput 3 mo, 18 ± 3; 24 mo, 32 ± 6; distal cauda, 3 mo, 16 ± 3; 24 mo, 12 ± 2, as representative sections) except for the corpus epididymidis. In that region, the number of cytotoxic T lymphocytes was significantly increased by 24 mo (3 mo; 23 ± 2; 24 mo, 45 ± 4; p < 0.001). It is of interest that in aged rats, accumulations of immunocompetent cells, mostly cytotoxic T lymphocytes and helper T lymphocytes, were often seen at the periphery of the epididymis, in the immediate proximity of blood or lymph vessels.
Effect of Luminal Content on the Number of Immune Cells in the Epididymal Epithelium and Interstitium
In Brown Norway rats of 18–24 mo of age, the number of monocytes-macrophages present in the epithelium was significantly higher in the initial segment and caput and corpus epididymidis when few spermatozoa were present in the epididymal lumen as compared to when numerous spermatozoa were found (Fig. 7A). In contrast, luminal content did not affect the number of monocytes-macrophages present in the epithelium of the proximal and distal cauda epididymidis. The quantity of spermatozoa in the lumen had no effect on the number of helper T lymphocytes (CD4+) present in the epididymal epithelium (Fig. 7B). Although the number of cytotoxic T lymphocytes (CD8+) was higher in the initial segment and caput epididymidis, this increase was not significant. In the corpus epididymidis of aged rats, luminal content significantly affected the number of cytotoxic T lymphocytes (Fig. 7C). In contrast, luminal composition had no effect on the number of cytotoxic T cells in the proximal and distal cauda epididymidis (Fig. 7C). Luminal composition did not affect the number of monocytes-macrophages, helper T lymphocytes, and cytotoxic T lymphocytes in the interstitium (data not shown).
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Effect of Age and Lumen Content on the Size of ED1-Positive Cells Present in the Epididymal Epithelium and Interstitium
In the epididymal epithelium of 3- and 12-mo-old rats, the overwhelming majority of ED1-positive cells was small (Fig. 8). In the initial segment and caput epididymidis, there was a significant increase by 18 mo in the proportion of large ED1-positive cells. This increase was accompanied by an increase in the proportion of medium and a relative decrease in the proportion of small ED1-positive cells. A similar trend was seen in the corpus by 18 mo and proximal cauda epididymidis by 24 mo. In contrast, age had no significant effect on the size of monocytes-macrophages in the distal cauda epididymidis. Furthermore, in aged rats, the proportion of large ED1-positive cells was significantly greater in the initial segment and corpus epididymidis when few spermatozoa were present as compared to when the lumen was filled with spermatozoa (Fig. 9). A similar trend was seen for the caput and proximal cauda epididymidis. Luminal composition had no effect on the size of ED1-positive cells in the distal corpus epididymidis. In addition, age and luminal composition had no significant effect on the size of ED1-positive cells present in the interstitial tissue (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monocytes-macrophages, helper T lymphocytes, and cytotoxic T lymphocytes were present in the epithelium at all ages. Our counting method allowed us to clarify the nature of halo cells. We confirmed that halo cells are immune cells and demonstrated that they comprise monocytes, helper T lymphocytes, and cytotoxic T lymphocytes. Furthermore we found a segment-specific increase with age in each of these immune cells in the epididymal epithelium. In addition, the incidence of immune cells was further enhanced in aged animals whose epididymal lumen contained few spermatozoa.
Identification of Immune Cells in the Epididymal Epithelium
Immune cells were present along the entire epididymal epithelium at all ages. Macrophages and T lymphocytes have been reported in both the epididymal interstitium and the epithelium under normal conditions (human [10, 23], rat [15, 24], mouse [25]). However, their precise origin is still unclear.
Yeung et al. [10] proposed that cells positive for an antibody against mature tissue-fixed macrophages were basal cells, based on their similar localization and morphological resemblance (human). In the present study, we demonstrated that in the Brown Norway rat, basal cells identified by an antibody against GST Yf do not recognize an antibody against monocytes-macrophages (Fig. 5). Our results are in agreement with the observation made by Flickinger et al. [15] that basal cells of the Lewis rat do not stain with an antibody against macrophages. Furthermore, under normal conditions, basal cells are elongated cells whereas ED1-positive cells display various shapes and sizes. In addition, we found that basal cells do not recognize antibodies directed against helper T lymphocytes, cytotoxic T lymphocytes, or B lymphocytes. Together, clearly these observations indicate that basal cells are not immune cells.
The nature of halo cells has been the subject of controversy since their discovery by Reid and Cleland in 1957 [3]. Halo cells are present throughout the epididymal epithelium and are usually defined as small cells with a narrow rim of clear cytoplasm [1]. Lymphocytes and monocytes have similar sizes, and their distinction based on nuclear morphology is at best difficult at the light microscope level. In the epididymal epithelium of young Brown Norway rats, the number of cells that stained for antibodies against monocytes-macrophages (ED1+), helper T lymphocytes (CD4+), and cytotoxic T lymphocytes (CD8+) was equivalent to the number of halo cells. In young Brown Norway rats, the vast majority of ED1-positive cells were of small or medium size. However, the proportion of large ED1 cells increased during old age. Interestingly, the combined number of ED1-positive cells is equivalent to the number of halo cells at 24 mo, if the large ED1 cells (most probably macrophages) are not entered in the equation. Thus, halo cells are composed of T lymphocytes and monocytes. This suggests that no or very few additional types of immune cells are resident or migrate into the epididymal epithelium of the Brown Norway rat under normal conditions. In aged rats, eosinophils [19] and B lymphocytes were occasionally seen in the epithelium. Thus, the occurrence of intra-epithelial migration of B lymphocytes, eosinophils, and, possibly, plasma cells, neutrophils, or basophils, is probably a rare event that occurs only under pathological conditions.
In the epithelium of young Brown Norway rats, T lymphocytes were the predominant immune cells. There appear to be species differences in the main types of immunocompetent cells present in the epididymal epithelium. In murine epididymis, macrophages are the most abundant immunocompetent cells [25]. However, as in Brown Norway rats, human epididymal immune cells are mainly composed of T lymphocytes, with the main lymphocyte subpopulation being the cytotoxic T cells [23, 30]. In Lewis and Wistar rats, helper T lymphocytes are present in the greatest relative abundance in the epididymal epithelium [15, 24].
Effects of Age on the Number of Immune Cells
An important finding of this study was the demonstration of a dramatic increase with advancing age in the numbers of each type of immune cell present in the epididymis. A higher number of lymphocytes and macrophages was reported in the epididymides of older men [16]. An increase of migratory immune cells with age has been reported in other tissues, such as the kidney (human [31, 32]). The trigger of this age-related immune response is presently unclear.
The increase in immune cells in the epididymis was segment-specific. Helper T lymphocytes and cytotoxic T lymphocytes were first recruited in the distal cauda epididymidis as early as 12 mo. This recruitment was probably due to subtle modifications of the epithelium since the appearance of the epithelium and lumen of the cauda epididymidis did not seem to differ from that of 3-mo-old rats [19]. The proportion of T lymphocytes present in the cauda epididymidis did not change significantly between 12 and 24 mo, and no major histological changes were reported in that segment at 12 mo or at later ages [19]. Furthermore, age had no effect on the incidence of monocytes-macrophages in the distal cauda epididymidis. The main effect of age on immune cells found in the distal cauda epididymidis was a doubling in the number of T lymphocytes. The distal cauda epididymidis appeared relatively less affected by aging for reasons that are not clear. In contrast to the distal cauda epididymidis, age had a major effect on the number of immune cells present in the other epididymal segments. By 18 mo, the number of monocytes-macrophages was dramatically increased in the initial segment, caput, and corpus. Thus, a new substrate or signals for monocytes-macrophages may emerge in these epididymal segments at 18 mo.
Despite a significant increase in the total number of monocytes-macrophages, helper T lymphocytes, and cytotoxic T lymphocytes present in the epithelium, the number of immunocompetent cells present in the peritubular interstitium was not markedly affected by age. Although there were few macrophages in the interstitial tissue at any age, there was a major increase in the proportion of macrophages in the epithelium of Brown Norway rats with age. Together these observations suggest that the substrate and/or signals for the migration of immune cells are not found in the interstitial tissue but rather in the epithelium.
In the epithelium, helper T lymphocytes, cytotoxic T lymphocytes, or monocytes-macrophages were always located between epithelial cells; this is in agreement with other studies [14–16]. Lymphocytes were frequently seen in the midst of the epithelium or near the tight junctions at all ages. Unlike helper T lymphocytes or cytotoxic T lymphocytes, monocytes-macrophages were always localized in the lower half of the epididymal epithelium, suggesting that their substrate might be different and located near the basement membrane. It is well established that the interaction between macrophages and lymphocytes is important for antigen uptake and processing [15, 32, 33]. Interestingly, in the epididymal epithelium, monocytes-macrophages and lymphocytes were often found in close proximity, suggesting an active interaction. Overall, a higher concentration of immune cells appeared to be correlated with major alterations of the epithelial structure, such as lysosome accumulation [19], suggesting that modification of the epithelial integrity is an important factor in the recruitment of lymphocytes. In addition, the dramatic accumulation of immune cells seen in old rats may create further damage to the epithelium.
In the young animal, the epithelium may contain enough immune cells to provide for its efficient protection and the prevention of autoimmune reaction against spermatozoa; the interstitial space may act as a reservoir of immune cells. Although a local proliferation of macrophages is possible [34], mitotic figures were not found in the epithelium or the interstitial space. However, an accumulation of helper T lymphocytes and cytotoxic T lymphocytes was often seen close to blood or lymph vessels located at the periphery of the epididymis. Thus, one may hypothesize that the interstitial reservoir may be insufficient to sustain the age-related increase in the demand for immune cells by the epithelium. Consequently, the interstitial space might serve as a pathway for the active recruitment of immune cells coming from the blood or lymph vessels into the epithelium.
Effect of Luminal Content on the Number of Immune Cells
In aged rats, there was a segment-specific positive correlation between the presence of an abnormal luminal content and the recruitment of immune cells in the epithelium. An increase in intra-epithelial lymphocytes and macrophages following vasectomy and sperm degeneration has been reported [35, 36]. In contrast, the number of intra-epithelial lymphocytes is lower in sexually immature rats than in the adult rat [14]. Interestingly, the production of spermatozoa begins at puberty, after the formation of the immune system tolerance to self-antigens (rat [6, 7]). Pachytene spermatocytes and later stages of spermatozoal development are identified as foreign components by the immune system (rat [37]). Thus, the breakdown product of spermatozoa or immature germ cells may be an antigenic stimulant [37–39]. Macrophages, helper T lymphocytes, and cytotoxic T lymphocytes may cooperate to prevent antigens from reaching the circulation, thereby creating an autoimmune response [15, 23, 30].
The effect of luminal content on the distribution of immune cells was segment-specific. The recruitment of cytotoxic T lymphocytes and macrophages was more marked in the initial segment and caput and corpus epididymidis of aged rats that had few spermatozoa in the lumen. Our results suggest that antigens of spermatozoal origin are present in these epididymal regions and, therefore, that the blood-epididymis barrier is likely to be leaky in aged animals.
In the young Brown Norway rat, a small number of helper T lymphocytes, cytotoxic T lymphocytes, and monocytes-macrophages were present in the epithelium. With increasing age, there was a major recruitment of immune cells into the epithelium. This recruitment was segment-specific and possibly related to various substrates (abnormal or "foreign" material) that accumulate with increasing age in the epithelium. The synergistic effect of an altered luminal content on the number of immune cells present in the epithelium suggests that some of these chemotactic factors or antigens are spermatozoa and/or the debris of immature germ cells leaking through an altered blood-epididymis barrier. The dramatic accumulation of immune cells in aged rats might be damaging to the epithelium and contribute to the senescence of the tissue.
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FOOTNOTES |
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2 Correspondence: B. Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, PQ, Canada H3G 1Y6. FAX: 514 398 7120; brobaire{at}pharma.mcgill.ca
Accepted: April 22, 1999.
Received: January 29, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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L. Marcon, B. F. Hales, and B. Robaire Reversibility of the Effects of Subchronic Exposure to the Cancer Chemotherapeutics Bleomycin, Etoposide, and Cisplatin on Spermatogenesis, Fertility, and Progeny Outcome in the Male Rat J Androl, July 1, 2008; 29(4): 408 - 417. [Abstract] [Full Text] [PDF]
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M. Carroll, M. Hamzeh, and B. Robaire Expression, Localization, and Regulation of Inhibitor of DNA Binding (Id) Proteins in the Rat Epididymis J Androl, March 1, 2006; 27(2): 212 - 224. [Abstract] [Full Text] [PDF]
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 L. Hermo, D. L. Chong, P. Moffatt, W. S. Sly, A. Waheed, and C. E. Smith Region- and Cell-specific Differences in the Distribution of Carbonic Anhydrases II, III, XII, and XIV in the Adult Rat Epididymis J. Histochem. Cytochem., June 1, 2005; 53(6): 699 - 713. [Abstract] [Full Text] [PDF]
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S. Seenundun and B. Robaire Cloning and Characterization of the 5{alpha}-Reductase Type 2 Promoter in the Rat Epididymis Biol Reprod, April 1, 2005; 72(4): 851 - 861. [Abstract] [Full Text] [PDF]
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 Y. Hu, Z. Zhou, C. Xu, Q. Shang, Y.-D. Zhang, and Y.-L. Zhang Androgen Down-Regulated and Region-Specific Expression of Germ Cell Nuclear Factor in Mouse Epididymis Endocrinology, April 1, 2003; 144(4): 1612 - 1619. [Abstract] [Full Text] [PDF]
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 E. Yakirevich, O. Yanai, Y. Sova, E. Sabo, A. Stein, J. Hiss, and M.B. Resnick Cytotoxic phenotype of intra-epithelial lymphocytes in normal and cryptorchid human testicular excurrent ducts Hum. Reprod., February 1, 2002; 17(2): 275 - 283. [Abstract] [Full Text] [PDF]
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