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Immunology |
Cooperative Reproductive Science Research Center, and Departments of Physiology and Microbiology and Immunology, Morehouse School of Medicine, Atlanta, Georgia 30310
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cytokines, environment, leptin, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The environmental signal that apparently cues the enhancement of immune function during the late summer and fall is a decreasing photoperiod [3, 7, 11, 12]. This survival mechanism is not limited to species that show a robust reproductive response to changing day-length. For example, the reproductive system of the house mouse is relatively insensitive to photoperiod, but this species shows photoperiod-induced fluctuations in the size of immune organs and immune function [13, 14]. Also, humans, like the house mouse and laboratory rat, do not show major seasonal fluctuations in reproductive function but do exhibit seasonal differences in immune system activity and disease prevalence [3].
The regulatory factors that mediate photoperiod-induced changes in immune status have not been fully identified. The effects of photoperiod length on the immune system appear to occur independent of reproductive status and sex steroids [3, 12]. Several humoral factors, including melatonin, have been proposed as potential mediators of the effects of photoperiod on immune function. Melatonin secretion is elevated during the nocturnal phase of the light-dark cycle, and this hormone has been shown to bolster immune function both in vivo and in vitro [3, 15, 16].
Another potential mediator of the effects of photoperiod on the immune system is leptin, a product of the obese gene that is produced predominantly by white adipose tissue [17, 18]. Mice that are homozygous for the mutation (ob/ob) of this gene have a leptin deficiency and are obese [19]. Leptin regulates appetite and energy expenditure at the hypothalamic level [19, 20]. However, it has become increasingly apparent that leptin exerts other direct effects on nonneural cells [21, 22]. The effect of leptin on immune function has recently received considerable attention [23, 24]. Both CD4+ and CD8+ T cells express the long isoform of the leptin receptor (Ob-Rb) [25], the form of the receptor that is involved in intracellular signaling [21]. The ob/ob (i.e., leptin-deficient) and db/db (i.e., deficient in the leptin receptor) mice have impaired T cell-dependent immunity [26, 27]. Humans with low body weight have reduced levels of leptin and a deficient cell-mediated immune response [28, 29]. Leptin apparently shifts the T-cell response to favor the production of proinflammatory cytokines (e.g., interleukin [IL]-2 and interferon-
) over cytokines that predominantly regulate humoral immune function (e.g., IL-4, IL-6, and IL-10) [30].
In many nonhuman species, the levels of adiposity change with the season; hence, seasonal fluctuations result in circulating leptin levels [31]. These seasonal rhythms are apparently driven by changes in photoperiod. Given that the data support a tropic role for leptin in immune function and that leptin secretion varies with the season, we investigated in the present study the involvement of leptin in mediating photoperiod-induced changes in immune function using the ob/ob male mouse as an animal model. A second objective was to assess the effect of leptin deficiency and photoperiod on testicular morphology and testosterone secretion, because photoperiod-induced changes in immune function may be mediated via altered gonadal function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Six-week-old ob/ob male mice (C57 BL/6J-Lep[ob]) and littermate controls (homozygous for leptin gene) were obtained from Jackson Laboratory (Bar Harbor, ME) and were housed under controlled laboratory conditions. All experiments were conducted according to the principles and procedures of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Fourteen ob/ob and 14 littermate control mice were housed under a 12L:12D photoperiod for 1 wk before the experiment. Animals were then divided randomly into four groups of seven animals each and were maintained under either a short photoperiod of 8L:16D (8:16) or a long photoperiod of 16L:8D (16:8) for a period of 10 wk. The four treatment groups of animals were as follows: 1) controls, 8:16; 2) ob/ob, 8:16; 3) control, 16:8; and 4) ob/ob, 16:8. The animals were fed a mouse formula diet (5008; Purina Mills, St. Louis, MO) ad libitum throughout the experiment, and daily food consumption and body weights were recorded for each mouse. At the end of 10 wk, animals were lightly anesthetized with halothane vapors and killed by decapitation. Trunk blood was collected for later assay of leptin and testosterone, and the thymus, spleen, epididymal fat pads, testes, and seminal vesicles were removed and weighed. The testes were placed in 10% formalin for later histological processing.
Spleens were cut into three to four small pieces and placed in chilled RPMI medium containing 2 mM L-glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10 µM 2-mercaptoethanol and were used immediately for isolation of splenocytes and to assess their proliferative response to the T-cell mitogen, concanavalin A (Con A).
Isolation of Splenocytes (Lymphocytes)
Lymphocytes were isolated as described previously [32]. Briefly, the spleen pieces were forced through a tea strainer into a Petri dish containing chilled medium. The cell suspension was filtered through nylon mesh and centrifuged at 1200 rpm for 10 min. The cells were then resuspended in 1 ml of a Tris-NH4Cl lysis buffer (pH 7.2) for 3 min to lyse any red blood cells. After the 3-min period, additional medium was added, and the cell suspension was centrifuged, resuspended in fresh medium, and washed three times. The cells were then resuspended in 1 ml of RPMI medium containing 2 mM L-glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 5% fetal calf serum, and the concentration of viable cells was determined by trypan blue exclusion. Cells were then diluted to a final concentration of 105 viable cells per 100 µl in the same media.
Lymphocyte Proliferation Assay
Lymphocytes (105 cells) were cultured for 48 h in duplicate 96-well culture plates with increasing concentrations of Con A (0, 0.6, 2.5, 5.0, 10, 20, and 40 µg/ml) at 37°C in a CO2 incubator. At the end of 48 h, culture supernatants were removed from one set of plates for the measurement of IL-2 (Th-1 proinflammatory cytokine) and IL-4 (Th-2 cytokine) production. The other set of plates was used to determine the level of lymphocyte proliferation via a colorimetric assay (CellTiter 96; Promega, Madison, WI). The maximum proliferation index for lymphocytes in response to the mitogen was calculated
for each animal.
Cytokine Assays
Production of IL-2 and IL-4 was measured using commercially available ELISA kits (Biosource International, Inc., Camarillo, CA). The minimum detection limits for IL-2 and Il-4 were 8 and 5 pg/ml, respectively.
Hormone Assays
Serum leptin and testosterone were measured using ELISA kits (Assay Designs, Inc., Ann Arbor, MI, and Alpha Diagnostic International, San Antonio, TX, respectively). The minimum detection limits for the leptin and testosterone assays were 5 and 10 pg/ml, respectively.
Histology
Testes were embedded in paraffin using conventional histological methods, and 8-µm sections were stained with hematoxylin-eosin for microscopic examination. We assessed testicular development and spermatogenesis by measuring seminiferous tubular area and determining the number of pachytene spermatocytes and round spermatids in each tubule and the percentage of seminiferous tubules exhibiting sperm bundles (elongated spermatids) using imaging technology (ImagePro Plus Software; Media Cybernetics, Silver Springs, MD). Twenty tubules were randomly selected from each animal (six animals/group) for these determinations.
Statistical Analysis
Two-way ANOVA was used initially to assess the potential significance of major effects (leptin deficiency and photoperiod). When the ANOVA indicated a significant interaction of the two variables (leptin deficiency and photoperiod), a Fisher Least Significant Difference (LSD) test was utilized for multiple comparisons of group means. Differences were considered to be statistically significant when P values were equal to or less than 0.05. All data are expressed as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of a leptin deficiency and photoperiod on body and organ weights are shown in Table 1. Body weights and the weights of epididymal fat pads in ob/ob animals were more than 2- and 5-fold greater, respectively, than in littermate controls (P < 0.0001 for both parameters). However, the testes and seminal vesicles of ob/ob mice were smaller than those of controls (based on ANOVA; main effect of leptin deficiency; P = 0.043 and 0.013, respectively). Photoperiod length did not affect body or reproductive organ weights. Weights of the thymus and spleen were not significantly affected by the leptin deficiency or photoperiod length (data not shown).
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Testicular morphology of ob/ob mice revealed a number of signs of impaired spermatogenesis (compare Fig. 1A from a control animal with Fig. 1, B and C, from two ob/ob mice). Within testicular sections of ob/ob animals were distinct regions where the seminiferous tubules exhibited reduced cellularity and an absence of elongated spermatids (Fig. 1B). Also, evidence was found of tubular vacuolization and condensation of germ cell nuclei (Fig. 1B, arrows), particularly in pachytene spermatocytes. Condensation of nuclei was also evident in the round spermatid population, which is characteristic of degenerating germ cells. A large number of syncytia formed by degenerating round spermatids were also observed in sections from the testes of ob/ob mice (Fig. 1C, arrows). In general, however, the effects of the leptin deficiency on testicular morphology were not diffuse. For example, large regions were observed within testicular sections of ob/ob animals where tubular morphology and spermatogenesis appeared to be normal (Fig. 1D). The patchiness of the effect of the leptin deficiency (by ANOVA) on testicular morphology was reflected in the marginal smaller seminiferous tubule areas (P = 0.078) and the marginally lower number of pachytene spermatocytes (P = 0.091) (data not shown). Photoperiod length did not affect the mean area of the seminiferous tubule or numbers of pachytene spermatocytes. However, photoperiod length did affect the percentage of tubules that exhibited sperm bundles (elongated spermatids, ANOVA, main effect of photoperiod, P = 0.037). The percentage of seminiferous tubules that exhibited sperm bundles from mice housed at 16:8 was lower than that of mice housed at 8:16.
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Serum Leptin and Testosterone Concentrations
Significant effects of the leptin deficient (P < 0.0001) and of photoperiod (P = 0.030) and a significant interaction (P = 0.031) of the two variables (based on the two-way ANOVA) on serum leptin concentrations (Fig. 2A) were found. Serum leptin values in ob/ob mice were near or below minimum detection limits regardless of the photoperiod conditions (Fig. 2A). However, in control mice, leptin levels were significantly higher (P = 0.030) in animals housed at 8:16 compared to those housed at 16:8. The tendency was for serum testosterone levels to be lower in ob/ob mice than in littermate controls, but because of the high variability of testosterone values in control mice, this difference did not reach the levels of significance (Fig. 2B). No effect of photoperiod on circulating testosterone concentrations was observed.
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Proliferative and Cytokine Responses of Lymphocytes to a T-Cell Mitogen In Vitro
Lymphocytes from ob/ob mice showed a subnormal (ANOVA, main effect of leptin deficiency, P = 0.017) maximum proliferation index in response to the mitogen, Con A (Fig. 3A). The cytokine production pattern by lymphocytes from ob/ob animals also differed from that of the cells of control mice. Production of the proinflammatory cytokine, IL-2, in response to Con A was subnormal (ANOVA, main effect of leptin deficiency, P = 0.0004) from cells of ob/ob mice irrespective of the photoperiod (Fig. 3B). Photoperiod length also affected (ANOVA, main effect of photoperiod, P = 0.027) IL-2 production by lymphocytes. Production of IL-2 by lymphocytes from mice housed at 16:8 was lower than that from cells of animals housed at 8:16.
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On the other hand, a leptin deficiency did not alter the production profiles of IL-4, a cytokine predominantly involved in the regulation of humoral immune responses (Fig. 3C). Lymphocytes from both the ob/ob and control animals produced similar amounts of IL-4 in response to Con A stimulation. However, IL-4 production from lymphocytes of mice (ob/ob and control) housed at 16:8 was reduced (ANOVA, main effect of photoperiod, P = 0.037) compared to that from the cells of animals housed at 8:16.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, the in vitro proliferative response of splenocytes from ob/ob mice to the mitogen, Con A, was subnormal, but photoperiod alone did not significantly alter this response. It should be noted that control mice housed under a short photoperiod (8:16) had significantly elevated leptin levels compared to controls housed under a long photoperiod (16:8). Thus, the data suggest that whereas leptin is necessary to maintain a normal lymphocyte proliferative response to this mitogen, photoperiod-induced changes in leptin secretion are not associated with significant alterations in this type of immune response to a foreign antigen.
Both leptin deficiency and photoperiod significantly altered cytokine production profiles by splenocytes in response to Con A, but these effects of the two variables appeared to occur largely independent of one another. For example, IL-2 production by splenocytes of ob/ob mice was reduced relative to that by cells of control mice regardless of the photoperiod conditions (ANOVA, main effect of leptin deficiency), and cells from mice housed under a short photoperiod produced more IL-2 than cells from animals housed under the long photoperiod (ANOVA, main effect of photoperiod). However, no significant interaction of the two variables on IL-2 production was found. The lack of a significant interaction suggests that the effects of the leptin deficiency and photoperiod on IL-2 production occurred independent of each other. Profiles of IL-4 production were not affected by the leptin deficiency, but cells from animals housed in the long photoperiod (ANOVA, main effect of photoperiod) exhibited an attenuated IL-4 response to the mitogen. These results indicate that both leptin and photoperiod have substantial effects on cytokine production profiles by lymphocytes, but the photoperiod-induced changes in these responses do not appear to be mediated by alterations in leptin secretion.
Leptin belongs structurally to the long-chain helical cytokine family that includes IL-2, IL-12, and growth hormone and that signals via a class I cytokine receptor [23, 33]. Leptin-deficient (ob/ob) mice exhibit a number of features usually associated with animals during chronic starvation, such as low body temperature, hyperphagia, infertility, and impaired immune function [23]. It has been proposed that leptin serves as an important link between nutritional status and T-lymphocyte function [23]. Both ob/ob and leptin receptor-deficient (db/db) mice have impaired T-cell immunity [26, 27], and a leptin-signaling deficiency compromises both humoral and cellular immunity and attenuates the symptoms associated with experimentally induced arthritis in mice [34]. Moreover, bacterial lipopolysaccharide administration to rodents up-regulates leptin expression in adipocytes and increases circulating leptin levels in addition to mobilizing defense responses [35, 36]. Leptin also protects mice from starvation-induced lymphoid tissue atrophy and increases thymic cellularity in ob/ob mice [37]. These data, along with evidence from the present study, support the idea of a major role for leptin in linking the level of nutrition to immune competence.
In the present study, splenocytes from ob/ob mice produced less of the proinflammatory cytokine, IL-2, but the leptin deficiency had no significant effect on production of the Th-2 prototype cytokine, IL-4. These data are in agreement with previous findings that leptin promotes the production of Th-1 cytokines but has an adverse effect on the production of Th-2 cytokines [30, 38]. Loffreda et al. [38] reported that leptin activates macrophages, induces their expression of proinflammatory cytokines, and increases macrophage function, whereas animals that are deficient in leptin signaling (ob/ob and db/db mice) exhibit impaired macrophage activity.
The effects of photoperiod on immune function vary according to the immunological parameter assessed and the animal model employed. In general, however, exposure of animals to a short photoperiod under laboratory conditions (abundance of food and mild temperatures) is associated with enhancement of immune status. The most frequently utilized mammalian models for these studies have been the deer mouse (Peromyscus maniculatus bairdii) and Siberian hamster (Phodopus sungoris), and results have varied considerably between these two species. The deer mouse is a temperate-zone animal, whereas the Siberian hamster is exposed to more severe shifts of environmental conditions in the wild. The differences in habitats may account for some of the differences reported for the two species. Under laboratory conditions, housing both species under a photoperiod that mimics winter day-length (8:16) is associated with testicular and reproductive tract regression [9, 10, 39, 40]. In deer mice, exposure to a short photoperiod at laboratory temperature results in an elevation of total serum immunoglobulin G levels and an enhancement of the lymphocyte proliferative response in vitro to Con A [9, 10]. In Siberian hamsters, basal proliferation of lymphocytes and natural killer cell activity were increased, but the humoral response to an antigen as well as granulocyte and monocyte phagocytosis of bacteria were attenuated under winter-like photoperiod conditions [39, 40]. Body weight, epididymal fat mass, and circulating leptin levels were reduced in hamsters housed under a short photoperiod, suggesting that leptin does not mediate photoperiod-induced enhancement of lymphocyte proliferation or natural killer cell activity but leaving open the possibility that leptin may be mediating the effects of photoperiod on humoral immunity in this species. These data appear to be in agreement with results from the present study in the mouse: Photoperiod has substantial effects on immune responses (e.g., cytokine production profiles) that occur independent of the status of leptin secretion (i.e., in both ob/ob and control mice).
Melatonin appears to play an important role in mediating the effects of photoperiod on immune function. Although secretion of this hormone rises during the dark phase of the light-dark cycle and lymphocytes from deer mice treated with melatonin exhibited an increased proliferative response to a T-cell mitogen, melatonin had no effect on antibody production in response to a foreign antigen [9, 41]. Because the effects of melatonin on lymphocyte proliferation occurred in both intact and castrated mice, melatonin-induced alterations in reproductive function did not mediate this response [41]. It would appear, then, that the photoperiod-induced changes in lymphocyte proliferation in the deer mouse and increased lymphocyte production of IL-2 and IL-4 from mice housed under a short photoperiod in the present study may be mediated by photoperiod-induced alterations in melatonin secretion. However, the pattern of melatonin secretion in response to photoperiod in the deer mouse, which is a seasonal breeder, may differ substantially from that in the house mouse, which is largely a nonseasonal breeder [13]; therefore, such a conclusion must be viewed with considerable caution.
It should be noted that in the present study, housing control mice under a short photoperiod elevated their serum leptin concentrations despite having no significant effect on body weight or epididymal fat pad mass (white adipose tissue). This result is suggestive that short photoperiod conditions increased the level of leptin expression in available white adipose tissue rather than increasing the level of adiposity in these animals. A comparison of leptin expression in adipose tissue between mice maintained under a short versus a long photoperiod would help to delineate whether this is true.
The present study provides further evidence that the effects of photoperiod on immune function are not mediated by photoperiod-induced changes in gonadal function. Photoperiod length had no significant effect on the weights of reproductive organs, testicular morphology, or serum testosterone levels in either ob/ob or control mice. However, a trend was observed for serum testosterone levels to be subnormal in ob/ob mice. Given that androgens are known to influence immune function [42], in a future study we may compare photoperiod-induced immune effects in intact versus castrated ob/ob mice. On the other hand, short-day enhancement of immune function in deer mice occurred in both intact and castrated animals [12].
A secondary objective of the present study was to further characterize the effects of leptin deficiency on testicular morphology and spermatogenesis. Testes and seminal vesicle weights in ob/ob mice were subnormal, and serum testosterone concentrations tended to be lower (but not significantly so) in ob/ob mice than in control mice. An examination of testicular morphology of ob/ob mice revealed marginally reduced seminiferous tubular size (area) and cellularity and subnormal numbers of spermatocytes. These findings are in agreement with those of previous studies reporting that the overall structure of the testes of ob/ob mice is abnormal and characterized by multinucleated spermatids, few spermatozoa, and a reduced amount of interstitial tissue [43, 44]. In the present study, the testes of ob/ob mice exhibited many premeiotic pachytene spermatocytes with darkly stained chromatin and eosinophilic cytoplasm, and evidence was also found for the presence of syncytia formed by, presumably, apoptotic round spermatids, a characteristic of degenerating germ cells [45]. What was perhaps somewhat surprising, however, was the less-than-diffuse effect of the leptin deficiency on testicular morphology. Despite the impaired nature of spermatogenesis in many tubules, large regions within the testes of the ob/ob mice showed testicular morphology and spermatogenesis that appeared to be normal.
In summary, the data from the present study do not provide definitive support for the contention that photoperiod-induced changes in leptin secretion mediate the effects of photoperiod on cell-mediated immune responses. Instead, both photoperiod length and leptin deficiency had significant, but largely independent, effects on the proliferative response and cytokine production profiles of lymphocytes. The results provide evidence for the complexity of the underlying processes by which decreasing photoperiod in the late summer and fall bolster immune system defense mechanisms in anticipation of the increased level of physiological stress and greater morbidity and mortality risks encountered by wild populations of rodents during the winter months.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: David R. Mann, Cooperative Reproductive Science Research Center, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310. FAX: 404 752 1056; mann{at}msm.edu
Received: 8 January 2003.
First decision: 13 January 2003.
Accepted: 3 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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