Characterization of Age- and Gender-related Changes in the Spleen and Thymus from Control Cynomolgus Macaques Used in Toxicity Studies

Abstract

Age- and gender-related lymphoid tissue variability in control male and female monkeys of various ages (under three years; three to six years; seven to fifteen years) was characterized. Spleen and thymus organ weights, organ-to-body and organ-to-brain ratios, morphology by light microscopy, and B- and T-cell immunohistochemistry (IHC) were evaluated. Splenic weights and ratios were not significantly different between various age groups or genders, except males and females in the three-to-six-years age group, who exhibited statistically significant changes from the under-three-years age group. No differences in the number of primary follicles, secondary follicles with germinal centers, B-cell follicles, and periarterial lymphoid sheath were seen between age groups or genders, and no trends were noted in the spleen. By IHC, no differences were observed in B- and T-cell splenic densities. Several age- and gender-related changes in weights and ratios were noted in the thymus. The thymus had a trend toward increased interlobular fat infiltration with increasing age in both males and females. Thymic delineation of the cortex and medulla was significantly decreased in the seven-to-fifteen-years age group for males only. The cortex-to-medulla ratio was significantly lower only in males in the seven-to-fifteen-years age group. B- and T-cell cellular density did not change across various ages.

Introduction

Immunotoxicity is a major consideration in the evaluation of drug safety because of the need to avoid the effects of drug-induced immunodeficiency, such as increased incidence of infections, neoplasia, and autoimmunity. Histological examination plays an essential role in identifying these effects on the immune system (Harleman 2000). At present, pathologists identify immunomodulatory effects via collective evaluation across a panel of parameters, including hematology, lymphocyte cellularity, organ weight changes, lymphoid organ morphology, incidence of infections, neoplasia or autoimmunity, and other immune function tests (Lappin and Black 2003).

Generally, immunosuppressive agents cause a decrease in lymphocyte cellularity, thymic depletion, and a concomitant decrease in organ size and weight (Gopinath 1996). However, owing to the dynamic nature of the lymphoid system and its response to various variables (i.e., stress, pregnancy or steroid hormone status, antigenic load, nutrition, and age), immunotoxicity and immunomodulation identification is a deceptive challenge, since not all morphologic or weight changes are indicative of a direct immunotoxic or immunomodulatory effect of a test article (Gopinath 1996).

Identification of immunotoxicity is further complicated by the scant description of histologic variability of the lymphoid organs in normal nonhuman primates, one of the most appropriate animal models for immunotoxicity. Rather, age-related changes in the thymus in humans are better characterized. Semiquantitative histologic variability in the human thymus indicates that thymic involution occurs earlier in males than females, and indicates the existence of a decreased number of Hassall’s corpuscles, with an increase in their size, a decreased delineation of the thymic cortex and medulla (corticomedullary junction) with increasing age, and an increased rate of thymic involution in response to disease (Smith and Ossa-Gomez 1981). Delineation of the cortex and medulla is typically used as a histologic marker of thymus maturity (Ewald and Walden 1988). In the mouse, for example, clear delineation of the cortex and medulla can be seen by gestational day 18, and it is delayed by conditions such as fetal alcohol syndrome (Ewald and Walden 1988).

Although the decreased delineation of the thymic corticomedullary junction was used in the study by Smith and Ossa-Gomez to grade an age-related change in humans, such a criterion is not typically used to grade and characterize an age-related change in other species such as nonhuman primates. Therefore, to characterize the age- and gender-related changes in lymphoid tissues in nonhuman primates, we have used the combination of tissue morphologic evaluation (corticomedullary junction delineation, interlobular fat infiltration, cortex-to-medulla ratio in the thymus, and number of primary and secondary follicles and periarterial lymphoid sheaths in the spleen) by light microscopy and T- and B-cell cellular density by immunohistochemistry. Normal slow physiologic age-related thymic involution should be distinguished from other types of involution that are related to stress (e.g., pregnancy, malnutrition, metabolic) or disease. Stress involution is usually mediated by the release of high levels of corticosteroids from the adrenal cortex (Schuurman et al. 1992). Microscopically, stress involution is usually characterized by the appearance of abundant numbers of starry-sky macrophages in the cortex followed by cortical lymphocyte depletion and shrinking of the thymic lobules with an increase in thickness of the interlobular septae resulting from fibrous tissue (van Baarlen et al. 1988).

The increased rate of thymic involution in response to disease is most likely a stress response and reversible. Acute thymic involution in infancy and childhood has been found to significantly correlate with the duration of illness (van Baarlen et al. 1988). In humans, the thymus also undergoes an age-associated involution that is irreversible (Pido-Lopez et al. 2001). The physiologic process of involution with age is usually gradual and progressive. In the early stages, it primarily consists of a decrease in the number of cortical lymphocytes with relative sparing of epithelial elements (Steinmann et al. 1985). In the advanced stages, the thymic parenchyma exhibits lymphoid depletion and is replaced by islands of epithelial cells with closely aggregated and cystic Hassall’s corpuscles and abundant adipose tissue. Simpson et al. (1975) found human thymic involution of the medulla to occur at a similar rate in males and females, but involuted at a slower rate than the cortex. More recently, Jayasharnkar et al. (2003) used flow cytometry and CBC lymphocyte counts in baboons to identify a significant correlation between increased age and B-cell decreases, and CD4, CD8, and T-cell increases. Using human peripheral blood mononuclear cells from healthy male and female volunteers, Pido-Lopez et al. (2001) found, using PCR, that signal joint T-cell receptor rearrangement excision circles (sjTRECs)—a measure of thymic output and, indirectly, size—progressively decrease with increasing age. Higher levels of sjTRECs and better thymic function were present in females than in males. A decrease in the number of newly generated CD4 or CD8 naïve cells (recent thymic emigrants) that survive thymic selection with increasing age is also gender related (Pido-Lopez et al. 2001). This finding corroborates similar findings that female thymuses involute more slowly than male counterparts. Similar studies on the spleen were not conducted.

Our study retrospectively analyzed thymuses and spleens from control cynomolgus monkeys to characterize the degree of variability in these lymphoid organs across various age groups and genders. We evaluated organ weights and ratios (organ-to-body and organ-to-brain ratios), tissue morphology by light microscopy, and quantification of B- and T-lymphocyte cellular density using immunohistochemistry.

Materials and Methods

Study Animals and Tissue Samples

Thymus and spleen tissue sections of control cynomolgus macaque (Macaca fascicularis) used in toxicity studies were obtained from the Ann Arbor Pathology Archives. Tissues from males and females were subdivided into three age groups based on sexual maturity (Table 1). The thymus and spleen were chosen, since the thymus is a primary lymphoid organ that is considered to be consistently sensitive to morphological effects (Harleman 2000), and the spleen is a secondary lymphoid organ. Additionally, these organs are found to have much less sampling variability than lymph nodes.

Organ Weights and Ratios

Thymus and spleen absolute weights and ratios from all monkeys were calculated relative to body and brain weights.

Histology and Lymphoid Tissue Morphology

Hematoxylin and eosin (H & E) slides of each section were examined by light microscopy. The following parameters were examined and determined by histology, immunohistochemistry, and/or image analysis:

Spleen: (a) B- and (b) T-cell cellular density in the white and red pulp, (c) number of primary and secondary follicles, and (d) the number of periarterial lymphoid sheaths (PALS); and

Thymus: (a) interlobular fat infiltration, (b) corticomedullary junction (delineation of cortex and medulla), (c) B- and (d) T-cell cellular density in the cortex and medulla, and (e) the cortex-to-medulla ratio.

The cortex-to-medulla ratio was determined based on the subjective relative thickness of the thymic cortex to the medulla as assessed by light microscopy.

Image Scanning

All thymus and spleen H & E sections were scanned using Nanozoomer imaging technology (Hamamatsu Inc., Japan, version 1.0, 2006).

Immunohistochemistry (IHC)

Three-micrometer sections were cut from formalin-fixed, paraffin-embedded blocks, mounted on positively charged glass slides, dried, deparaffinized in three changes of xylene for two minutes each, and then rehydrated through graded ETOH rinses to inactivate endogenous peroxidase activity, including hydrogen peroxide solution. Sections were stained using a Ventana Discovery XT stainer (Ventana Medical Systems, Tucson, AZ, USA). Monoclonal antibodies raised against human CD3 at 1:500 and CD20 at 1:500 dilutions were used to identify T- and B-cells, respectively. Sections were then subjected to heat-induced epitope retrieval (HIER). Sections were incubated with the primary antibody for thirty minutes at 37°C, followed by anti-mouse biotinylated secondary antibody at a 1:1000 dilution for thirty minutes at 37°C. Streptavidin-HRP was then applied to the sections, and diaminobenzidine (DAB) was used as the chromagenic enzymatic substrate. Finally, the sections were counterstained with hematoxylin, dehydrated, and coverslipped.

Data Analysis

A four-point semiquantitative analysis was used to grade the tissues. The grading scale was delineated as: (1/+) = minimal; (2/++) = mild; (3/+++) = moderate; and (4/++++) = marked. Thymus and spleen immunostaining were evaluated for parameters previously described in the histopathology section.

Statistical Analysis

Age groups and gender were compared using two-way analysis of variance (ANOVA) with interaction. If the interaction between age group and gender was significant, then one-way ANOVA was used within age group and sex. In the case where the data did not follow the normality assumption, Kruskal-Wallis (for age group) and Mann-Whitney (for gender) one-way ANOVA on ranks was used.

Results

Organ Weights and Ratios

Organ weights and ratios were graphed against increasing age (Figures 1 and 2). The range, mean, and standard deviation for organ weights and ratios for each age group and gender are shown in Tables 2 and 3. The range and mean of absolute spleen weights were generally larger than those of females across various age groups; however, this finding was not statistically significant. The range and mean of absolute spleen weight were not statistically significant between age groups of the same gender and across genders (except in the three-to-six-years age group of males and females). Absolute spleen weights were statistically and significantly smaller only in females when compared to males in the three-to-six-years age group. The range and mean of spleen-to-body weight ratio was not statistically significant between age groups of the same gender (except in males and females from the three-to-six-years age group) and across genders. The range and mean of spleen-to-body weight ratio in the three- to-six-years age group was significantly smaller within males or females when compared to the under-three-years age group, but not from the seven-to-fifteen-years age group. The range and mean of spleen-to-brain ratio was not statistically significant between age groups of the same gender and across genders (except in the three-to-six-years age group of males or females). The range and mean of spleen-to-brain ratio in the three-to-six-years age group was significantly smaller in females when compared to males.

The range and mean of absolute thymus weight was statistically significant between age groups of the same gender (except males or females in the under-three-years age group and in females in the seven-to-fifteen-years age group) and across genders (except in males or females in the under-three-years age group). The range and mean of absolute thymus weights were generally getting smaller and at a faster pace with increasing age in males compared to females. However, absolute thymus weight of males in the under-three-years age group was statistically and significantly larger when compared to that of females. Absolute thymus weight of females in the seven-to-fifteen-years age group was statistically and significantly larger when compared to that of males. Absolute thymic weights in the three-to-six-years age group were not statistically significant when compared between males and females. In males, absolute thymic weights from the three-to-six-years age group were significantly smaller than those thymuses from the under-three-years age group and significantly larger than thymuses from the seven-to-fifteen-years age group. In females, on the other hand, absolute thymic weights from the three-to-six-years age group were significantly smaller than thymuses from the under-three-years age group, but not from the seven-to-fifteen-years age group.

The range and mean of thymus-to-body ratio was statistically significant between age groups of the same gender (except in males or females in the under-three-years age group and in females in the seven-to-fifteen-years age group) and across genders (except in males or females in the three-to-six-years and under-three-years age groups). In males in the three-to-six-years age group, the range and mean of thymus-to-body ratio was significantly smaller when compared to the under-three-years age group and significantly larger when compared to the seven-to-fifteen-years age group. In females in the three-to-six-years age group, on the other hand, this ratio was significantly smaller when compared to the under-three-years age group, but not when compared to the seven-to-fifteen-years age group. When this ratio was compared across genders, it was statistically and significantly smaller only in males of the seven-to-fifteen-years age group.

The range and mean of thymus-to-brain ratio was statistically significant between age groups of the same gender (except in males or females in the under-three-years age group and females in the seven-to-fifteen-years age group) and across genders (except in males or females in the under-three-years age group). Thymus-to-brain ratio of males in the under-three-years age group was statistically and significantly larger when compared to females. This ratio in females of the seven-to-fifteen-years age group was statistically and significantly larger when compared to males. The three-to-six-years age group thymus-to-brain ratio was not statistically significant when compared between males and females. Males in the three-to-six-years age group had significantly smaller ratios than those from the under-three-years age group and significantly larger ratios from those from seven-to-fifteen-years age group. In females, on the other hand, the thymus-to-brain ratios from the three-to-six-years age group were significantly smaller than those from the under-three-years age group, but not from the seven-to-fifteen-years age group.

Histology, Lymphoid Tissue Morphology, and IHC

Histological observations made from H & E by the original study pathologists are shown in Tables 4 and 5. Lymphoid hyperplasia of the spleen was not significantly different across age groups within the same gender and between genders. Incidences of minimal lymphoid hyperplasia were 15% in males from the under-three-years age group and minimal (10%) to mild (5%) in the three-to-six-years age group. Splenic lymphoid hyperplasia was not diagnosed in males from the seven-to-fifteen-years age group. In females, minimal (10%) to mild (10%) lymphoid hyperplasia was diagnosed in the under-three-years and seven-to-fifteen-years age groups. The female three-to-six-years age group had minimal (10%) and mild (5%) lymphoid hyperplasia. There were occurrences (5%) of congestion in the male three-to-six-years age group and foamy macrophage infiltrates (5%) of female spleens. For the thymus, the percentage of involution increased with increasing age in males from the under-three-years age group (5%–10%), three-to-six-years age group (5%–15%) to the seven-to-fifteen-years age group (20%–60%). In females, the percentage involuted was 5%–10% in the three-to-six-years age group and 10%–20% in the seven-to-fifteen-years age group. This finding supports the trend toward decreasing thymus weights and ratios with increasing age. Focal to multifocal thymic cysts occurred in both males and females. A small percentage of congestion (5%) was reported in females of the seven-to-fifteen-years age group. In males of the under-three-years age group, 20% of esosinophilic infiltrates was reported.

The spleen and thymus cellular components were evaluated semiquantitatively (four-point scale) and quantitatively. Splenic cellular components that were evaluated included white and red pulp B- and T-cells, number of primary and secondary follicles, and PALS. Thymic cellular components included interlobular fat infiltration; corticomedullary junction (delineation of cortex and medulla); medullary and cortical B- and T-cells; and the cortical-to-medullar ratio. An example of splenic primary follicles, secondary follicles with germinal centers, PALS, and B- and T-cell cellular density from a young (Figure 3A) and a more mature (Figure 3B) monkey are shown in Figure 3. An example of thymic delineation of the cortex and medulla, interlobular fat infiltration, and B- and T-cell cellular density from a young (Figure 4A) and a more mature (Figure 4B) monkey are shown in Figure 4. The number of primary follicles, secondary follicles with germinal centers, B-cell follicles, and PALS did not change in the spleen between age groups or genders, and no trends were evident (Figure 5).

Thymic interlobular fat infiltration was significantly lower in the youngest (under three years) age group than the three-to-six or seven-to-fifteen-years age groups for both males and females (Figure 6). A trend toward increased interlobular fat infiltration with increasing age in both males and females was evident. Thymic delineation of the cortex and medulla was significantly decreased in the seven-to-fifteen-years age group for the males, but not for the females (Figure 6). The cortex-to-medulla ratio was significantly lower in males in the seven-to-fifteen-years age group, but not females (Figure 6). Semiquantitative IHC evaluation of B- and T-cell densities in the spleen (T-cell density in PALS and red pulp and B-cell density in follicles and red pulp) and thymus (cortical T-cells, medulla T- and B-cells) of males revealed no significant differences in T- and B-cell densities in various microanatomic compartments.

Discussion

For the spleen, absolute weights and ratios were not statistically significant between age groups of the same gender and across genders (except in the three-to-six-years age group of males and females). However, the range and mean absolute splenic weights, although not statistically significant, were generally lower in females than in males, and no significant differences in splenic congestion between genders was noted in our study. This finding is consistent with what has been seen in normal male and female spleens from humans (Sprogøe-Jakobsen and Sprogøe-Jakobsen 1997). However, this higher male splenic weight in humans did not correlate with gender or age, and no significant differences in the degree of congestion were noted between genders (Sprogøe-Jakobsen and Sprogøe-Jakobsen 1997).

In our study, no statistically significant differences in the range and mean of spleen-to-body ratio between age groups of the same gender (except in the three-to-six-years age group males or females) and across genders were noted. In humans, the weight of the spleen correlated positively with body weight (Sprogøe-Jakobsen and Sprogøe-Jacobsen 1997). In one study in Beagle dogs, spleens from one day to eleven months of age were weighed and compared with body weights. It was found that the weight of the spleen increased drastically at and after two months of age; however, the spleen-to-body weight ratio remained the same at two months of age and decreased afterward (Yang and Gawlak 1989). In Fischer 344 rats, a linear increase in splenic weight with age between four and thirty months of age was reported (Cheung and Nadakavukaren 1983). However, fewer cells were recovered from these rats with increasing age (Cheung and Nadakavukaren 1983). No significant differences in the number of PALS or follicles or in B- and T-cell cellular density with increasing age or between genders were noted. Percentage area of splenic tissue staining for B- and T-cells was determined by a virtual microscopic (Definiens) image analysis method (data not shown). These preliminary data support these findings, since their percentage area did not change significantly with increasing age and between genders.

Collectively, these results suggest that splenic changes are a result of factors other than age or gender. Because of the lack of studies, it is hard to compare these results to other species. Jayasharkar et al. (2003) did note a decrease in the relative numbers of B-cells and an increase in the relative number of T-cells in whole blood with increasing age in baboons. In humans, an age-related decrease in splenic T-cells in the T-cell zones, but not those T-cells that occupy splenic B-cell areas, was observed (Banerjee et al. 2000).

In the thymus, several age- and gender-related changes were noted. Age-related changes are likely attributable to physiologic involution that occurs with increasing age. Involution explains the decrease in thymus weight and in thymus-to-body and thymus-to-brain ratios, increased interlobular fat infiltration, decreased corticomedullary delineation, and decreased cortex-to-medulla ratio. There were no differences in the B- and T-cell densities in the thymus, a finding supported by other studies. For example, Smith et al. looked at fifty control human thymuses and found a decrease in the delineation of the cortex and medulla and an increase in adipose tissue (Smith and Ossa-Gomez 1981). Also similar to our findings, there were no differences in B- or T-cells in the cortex or medulla for males. However, lymphocyte density did decrease in human females in the fifth and sixth decade. This disparity may have been owing to the nominal sample number (n = 2 and n = 4 for respective decades) in the Smith study. This difference between female humans and nonhuman primates is hypothesized to be age of menopause onset. In nonhuman primates, menopause begins between twenty-four and twenty-nine years (Gilardi et al. 1997), whereas the average onset in humans is fifty-one years. In our study, the oldest female population had a mean age of 10.4 years, and the oldest female was 13.7 years of age. Thus, there was no correlation between menopausal humans and decreased thymic lymphocytes in older women that would not be expected in the older female monkeys studied.

For gender differences in the thymus, it is interesting that females do not experience thymic changes to the same degree as males. This gender-related difference is most likely owing to hormonal differences between genders. Male and female thymic weights and ratios decreased with increasing age, with males decreasing more rapidly. In our oldest group (seven to fifteen years), male thymus weight and ratios were even significantly lower than females. This gender difference has also been reported in human studies by Smith and Ossa-Gomez (1981), who found that females had less interlobular fat than males in the first four decades of life, but had an equivalent amount by the fifth and sixth decade, after menopause. Results presented here suggest that males have more interlobular fat infiltration than females, and that females did not have a similar amount of interlobular fat infiltration in the oldest age group.

Discrepancy in our results is probably because the female nonhuman primate population in this study had not yet undergone menopause. We would expect the female thymuses to look more like the male thymus once they have undergone menopause. In addition, we used fewer monkeys in the older age group in our study because typically most monkeys used in the pharmaceutical industry and toxicity studies are juveniles or young adults. Therefore, it could be that studying a larger number of the older monkeys would explain this discrepancy.

Another factor that may explain gender-related differences in the thymus may be related to stress. Significant changes in stress response gene expression in the thymus were observed between male and female mice (Lustig et al. 2007). Stress-induced thymic involution was higher in female mice than males (Dominguez-Gerpe and Rey-Menez 1998).

Organ weights have been historically considered an unreliable measure of drug-induced changes. As a result, organ-to-body and organ-to-brain ratios are frequently used to reduce variability because of differences in body size and weight and lean body mass. A high level of variability was noted in our data, especially in the thymus. The splenic range of variability in absolute weight, spleen-to-body, and spleen-to-brain ratios was approximately four- to five-fold and three- to four-fold in both the under-three-years and three-to-six-years age groups in males and females, respectively. The range in the seven-to-fifteen-years age group was two-fold and two- to three-fold in males and females, respectively. Significant alterations in splenic weights can occur if the spleen is congested and contracted, which can lead to significant changes in splenic ratios. The thymic range of variability in absolute weight, thymus-to-body, and thymus-to-brain ratios was approximately seventeen-to twenty-nine-fold and three- to five-fold in the under-three-years age groups in males and females, respectively. The range in the three-to-six-years age group was approximately five- to eleven-fold and three- to four-fold in males and females, respectively. The range in the seven-to-fifteen-years age group was approximately five- to six-fold and three- to four-fold in males and females, respectively.

Thymic weight is also complicated by its physiologic involution, which occurs with increasing age. In addition, the thymus is sensitive to stress, which leads to glucocorticoid-induced thymocyte apoptosis and subsequent thymic involution (Tarcic et al. 1998). It would be interesting to compare the control ranges to ranges for spleens and thymuses from monkeys treated with immunostimulatory and immunosuppressive drugs to see if one can distinguish between treated and control nonhuman primates, especially since the thymus is the first lymphoid organ to respond to immunotoxic compounds and does so with a decrease in size and weight and with T-cell depletion of the cortex (Kuper et al. 2000).

Potential future studies include: (1) compare current results to virtual microscopic (Definiens) quantitative image analysis results; (2) determine ability to distinguish spleens and thymuses treated with an immunotoxic compound from controls, using the data presented and in conjunction with Definiens image analysis; (3) evaluate the relationship within the study group and of spleen and thymus B- and T-cell densities; and (4) evaluate the number of Hassall’s corpuscles and the size of white pulp and PALS in the spleen.