INTRODUCTION

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ADAPTATIONS IN PHYSIOLOGY and behavior are strategies that nontropical species use to adjust to seasonal changes in metabolic fuel and inclement conditions. Breeding, molting, migration, and other energetically expensive activities are synchronized to coincide with the availability of food or favorable local conditions; these typically occur during the long days between the spring and fall equinoxes (8, 40). However, retrenchment of particular physiological functions during winter, e.g., hibernation or torpor, does not necessarily ensure survival. Seasonal cycles of illness and death occur among many populations of animals and humans. In temperate and boreal regions, animals die most frequently during the winter (22, 27), a possible consequence of exposure to stressors that enhance the risk of infection and opportunistic diseases. These stressors may include, but are not limited to, decreased quality and quantity of nutrition, reduced temperatures, increased competition from conspecifics, lack of protective cover, and predation.

Although seasonal incidences of disease and death are natural parts of the life cycle, animals do successfully adapt to environmental challenges. The annual change in photoperiod is the most reliable proximate cue that predicts seasonal challenges in climate, nutrition, and opportunistic pathogens. Day length and direction of change each day precisely signifies the time of year and predicts the coming season. The importance of this environmental cue is the fact that photoperiod regulates seasonal breeding and reproductive function in a variety of species (18, 21); births typically are timed to coincide with favorable springtime conditions. Other environment considerations, e.g., temperature or nutrients, can modulate physiological function but they are of limited value to forecast changes in season. Therefore it is reasonable to suggest that animals have developed the ability to use photoperiod information to forecast recurrent conditions associated with impending changes in the seasonal environment.

Adaptations in immune system function present one strategy that may promote individual survival in relation to a seasonal incidence of opportunistic diseases or changes in environmental conditions. Seasonal changes in disease prevalence and immune function are well known among humans: a pronounced influenza and cold season occurs during winter along with increases in malaria, dysentery, measles, asthma, arthritis, and many forms of cancer (6, 32). Evidence suggests seasonal changes in immune function by the host rather than in the parasite or pathogen are a possible explanation. However, field study results may be influenced by a complex variety of environmental conditions that may acutely or transiently affect immune function. Despite potential confounds, seasonal fluctuations in innate and acquired immune functions have been documented (43). Measures of immune cell counts, lymphoid organ weights, or T cell-dependent antibody responses to xenogeneic antigens are generally enhanced in winter. Moreover, laboratory experiments in which only photoperiod is manipulated indicate that exposure to short days increased mass of the spleen and thymus, as well as enhanced numbers of lymphocytes and neutrophils (reviewed in Ref. 31). These data implicate short days as possibly enhancing functional capabilities by lymphoid and myeloid cells. Although direct measures of functions by distinct immune cell populations have not been extensively studied, tumorigenesis was reduced while basal lymphocyte proliferation or mitogen-induced splenocyte proliferation were potentiated in rodents in short days (11, 14, 30).

Based on the possibility that immune cell function is generally enhanced by exposure to short days, the present report tested the hypothesis that distinct immune cell functions are influenced by ambient photoperiod. Aspects of innate (phagocyte activity and natural killer cell cytotoxicity) and acquired (spontaneous lymphocyte proliferation) immune function were studied in hamsters in long or short days. The Siberian hamster was chosen as the animal model because the neuroendocrine mechanism that mediates photoperiod control of reproduction has been studied extensively in this species (5, 41, 42). Findings from this study indicate that profound but selective effects on immune functions are associated with the prevailing photoperiod.

	METHODS

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Male Siberian hamsters (Phodopus sungorus, also known as Djungarian; 4-10 mo of age) were born and reared in long days (16 h of light from 1800-1000 PST). Temperature and humidity in the two adjacent vivarium rooms were held constant (22°C and 45%); food and water were continuously available. The vivarium is certified by the American Association of Accreditation of Laboratory Animal Care, and all procedures were approved by the institutional animal research committee. For this study adult hamsters remained in long days or were in short days for 4-6 wk (8 h of light from 0200-1000 PST). For immune cell function tests about 0.5 ml of whole blood was drained from the ocular sinus of each hamster with a heparinized microcapillary tube into a 12 × 75 mm test tube or a 2.5-ml microcentrifuge tube that contained 0.1 ml of heparin (1,000 U/ml). All blood samples were taken 2-3 h before lights off. When splenocytes were required (see Natural killer cell cytotoxicity assay), hamsters were killed 2-3 h before lights off. Studies were conducted between the autumnal and vernal equinoxes.

Assessment of Immune Cell Function

Phagocytosis and oxidative burst activity. Granulocytes and monocytes in erythrocyte-lyzed whole blood from hamsters in long or short days (0.1 ml) were segregated by FACSort flow cytometry according to previously described methods (33, 39). To quantify phagocytosis, 0.1 ml of whole blood (in duplicate) was incubated with FITC-labeled Staphylococcus aureus bacteria (on average 117/phagocyte, 1 h in the dark at 37°C); internalization (phagocytosis) of FITC-labeled bacteria by granulocytes and monocytes was quantified by flow cytometry. Data are expressed as the mean fluorescence channel number for each FITC-positive phagocyte population. To assess oxidative burst activity by phagocytes, 0.1 ml of whole blood was incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA, 0.5 mM) for 1 h at 37°C in the dark in the absence of (basal) or with Staphylococcus aureus bacteria (stimulated). In this assay intracellular conversion of DCF-DA to a fluorescent molecule occurs within phagocytes when a reactive oxygen radical is encountered. As an indication of oxidative burst activity, the relative fluorescent intensity (fluorescent channel) of granulocytes and monocytes was evaluated by FACSort flow cytometer and expressed as the log mean channel fluorescence.

Natural killer cell cytotoxicity assay. Splenocytes from hamsters in long or short days were isolated by Ficoll gradient centrifugation (7). Duplicate aliquots of cells (1 × 10⁶) were incubated with YAC-1 target cells (American Type Culture Collection, Rockville, MD) in effector-to-target cell ratios that ranged from 5:1 to 40:1. A two-color flow cytometry method was employed in which live DiO-labeled target cells were distinguished from dying or dead cells that had become stained with propidium iodide (12, 36). Debris thresholds were set for each sample run. Cytotoxicity was normalized to a hamster spleen cell pool as a quality control for each run and reported as lytic units (9).

Spontaneous blastogenesis. This test assesses basal lymphocyte proliferation measured either in whole blood or purified lymphocyte preparations (7). Basal proliferation and differentiation by immune cells in vivo are reflected by in vitro DNA synthesis, i.e., [³H]thymidine uptake (34). The assay followed standard clinical laboratory procedures (15). Aliquots of whole blood (0.05 ml in triplicate, n = 5) from males in long or short days were incubated with 1 µCi [³H]thymidine and 0.01 ml RPMI 1640 culture medium in a microculture plate for 2 h at 37°C in humidified 5% CO₂ (29). Cells were harvested onto glass fiber filter mats, washed, and [³H]thymidine incorporation was measured using an aqueous scintillation cocktail (Packard, Garden Grove, IL). In addition, lymphocytes were isolated from whole blood (0.2 ml) by Ficoll-Hypaque density gradient centrifugation, washed, and resuspended at 1 × 10⁵ cell/well in RPMI 1640 medium with 10% fetal calf serum. After resting the cells for 2 h at 4°C, aliquots of 1 × 10⁵ cells (0.1 ml in triplicate) with 1 µCi [³H]thymidine were incubated for 12 h at 37°C (humidified CO₂) and [³H]thymidine incorporation measured as previously described (29).

Statistics. Data were log transformed to normalize variance. A t-test was used for natural killer cell data. For other tests data were evaluated by one-way ANOVA; individual comparisons were made with Bonferroni's multiple range test. Levene's test was used to determine homogeneity of variance. P < 0.05 was considered significant for these analyses.

	RESULTS

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After 4-6 wk of short days hamsters had palpable small testes. Postmortem inspection of the gonads indicated that testes were regressed in males in short days (<100 mg) but were large in hamsters in long days (within a range of 600-900 mg). Mean body weights ranged from 32 to 36 g and were not significantly different in hamsters in short or long days.

Phagocytosis and Oxidative Burst Activity

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Phagocytosis and oxidative activity in granulocytes (GRAN) and monocytes (MONO) from hamsters in long (LD) or short (SD) days. Data are the mean fluorescence channel number (±SE, n = 6 each) for each FITC-positive phagocyte population. Fluorescence indicates uptake of FITC-labeled Staphyloccocus aureus bacteria by respective phagocytic cells. Oxidative burst is indicated by the mean fluorescence channel number for each dichlorodihydrofluorescein-positive phagocyte population following no treatment (BASAL) or after stimulation (STIM) with Staphyloccocus aureus bacteria in culture. Assay details are discussed in METHODS. * P < 0.01 vs. LD group (ANOVA), a P < 0.05 vs. BASAL activity same photoperiod.

Natural Killer Cell Cytotoxicity

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of exposure to long days (LD = 16 h of light daily, n = 23) or short days (SD = 10 h of light daily for >4 wk, n = 19) on nonspecific cytolytic natural killer cell activity in the hamster. Lytic units (means ± SE) were calculated based on killed cells relative to effector-to-target cell ratios as previously described (14, 16). * P < 0.01 vs. LD group (t-test).

Spontaneous Blastogenesis

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Incorporation of [3H]thymidine by cells in whole blood or by sorted lymphocytes from hamsters in long days (LD) or short days (SD). Data are means ± SE (n = 5 each). * P <0.05 vs. LD group (ANOVA). See METHODS for experimental details.

	DISCUSSION

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Seasonal biological function commonly indicates a role for photoperiod as a mediator in physiological adaptations to changes in the environment. Photoperiod control of seasonal reproduction is a significant example (8, 18). Reports that immune cell numbers and immunoglobulin concentrations vary with respect to season or day length (reviewed in Ref. 32) raise the possibility that photoperiod may also influence functional capabilities of immune cells. The contribution of the present study is the finding that changes in function by certain immune cells were associated with exposure to short days. The perspective that short days generally promote immune system function proves to be simplistic. Rather, short days selectively enhance natural killer cell activity and basal spontaneous proliferation. By contrast, phagocytic and oxidative burst activity, particularly by granulocytes, are reduced in hamsters in short versus long days.

As a component of the innate immune system, natural killer cells serve a complex role in a rapid, front-line response cascade to "foreign" invasion (30). Natural killer cells protect against infection and help to discriminate "non-self" from "self." These cells subsequently contribute to immune activation by secreting cytokines that modulate aspects of both innate and acquired immunity. Thus natural killer cells defend against opportunistic disease and tumorigenesis. The short-day-associated increase in natural killer cell activity cannot be attributed to changes in cell numbers because the response was based on lytic units, a calculation dependent on known effector-to-target cell ratios. Rather, enhanced activity by this lymphocyte subset from hamsters in short days may reflect in vivo activation, perhaps by increased production of inflammatory cytokines, i.e., by interferon- and/or interleukin-2 from natural killer cells themselves or T helper 1 lymphocytes (20) or by interleukin-12 from macrophages (38).

The possibility that in vivo basal immune cell function is enhanced in hamsters in short days is supported by findings for enhanced spontaneous blastogenesis in whole blood, a general indication of both innate and acquired immune cell activities in circulation. Proliferation by leukocytes and, in particular, lymphocytes were found to be greater in hamsters in short days compared with that in long days. As a reflection of basal activation in vivo, enhanced proliferation in this assay may reflect clonal expansion by T and B cells, as preparatory for primary immune responses and/or expansion of memory cell clones, as a secondary immune response to opportunistic pathogens. Because resources are more limited in winter than at other times of the year, augmentation in basal activity by some lymphocyte populations in hamsters in short days may ensure an accelerated response to infection through activation of memory cells.

The adaptive value for selective, rapid, and robust immune responses in animals in short compared with long days may be to more effectively mitigate the spread of infection that could threaten survival. In fact, seasonality in mitogen-induced lymphocyte proliferation (primary acquired immune response) is suggested in the mouse; a winter nadir is followed by a springtime peak (35). However, studies of adaptive responses to photoperiod are limited in some inbred laboratory species, mice and rats in particular. By example, many strains of inbred mice have a genetic defect that precludes pineal melatonin synthesis (19), a crucial element in the neuroendocrine mechanism that mediates many of the effects of photoperiod on physiological function. In the cotton rat, a rodent that is highly photoperiodic, seasonality in lymphocyte proliferation in response to antigen were comparable to that in the mouse, i.e., December nadir and August peak (23). Therefore, further study is needed in species in which photoperiod control of physiological adaptive responses have been established to support the contention that short days enhance basal and pathogen-induced lymphocyte function.

Whether photoperiod contributes to an overall strategy by the host immune system to meet seasonal opportunistic challenges is further suggested by evidence that phagocytes (especially granulocytes) from hamsters in short days have a reduced basal capacity to identify and respond to pathogens compared with those in hamsters in long days. A blunted basal oxidative burst response by phagocytes from animals in short days is consistent with the expectation that hamsters are likely to be exposed to fewer bacterial pathogens during the short days of winter compared with the summer. Phagocytosis by monocytes, a likely reflection of macrophage processing and presentation, is a key initial step in processing foreign material for antigen presentation to T cells (17). This function was not tested in the present study. Thus primary acquired immune response capabilities by these cells may be compromised in hamsters in short days. By contrast, an increased capacity to process and present antigens during the long days of summer may coincide with breeding activity and more diverse conspecific interactions in the environment. Strong primary responses by hamsters in long days may include proliferation of clones of specific memory cells; the rapid recall of such clones would enhance immune response capabilities during the short days of winter.

The present findings provide the foundation to determine whether the effects of photoperiod on immune cell functions may be influenced by the same neuroendocrine mechanism that mediates photoperiodic control of reproduction. The pineal melatonin rhythm transduces information about day length to regulate the hypothalamic gonadotropin-releasing hormone pulse generator and gonadotropin secretion (3, 5, 10, 21). In this system the extended duration of the nighttime melatonin rise is the critical signal that suppresses neuroendocrine secretion and leads to gonadal atrophy. Thus in the present study, short-day-induced testes regression cannot be separated from observed changes in immune cell function. Reduction in androgens in the circulation of short-day hamsters may promote selective lymphocyte functions because testosterone has suppressive effects on cell-mediated immunity (37). As such, control of immune cell activity by photoperiod cannot exclude a role for gonadal steroids in mediating immune system consequences associated with exposure to short days.

Another neural pathway in which the melatonin may indirectly modulate immune cell functions could involve prolactin and glucocorticoid secretion. In a variety of species, prolactin is increased by exposure to long days and the associated short-duration melatonin rhythm, compared with that in animals in short days (reviewed in Ref. 31). Increased prolactin in circulation may have promoted phagocytosis and oxidative burst activity in the present study, a finding analogous to that previously found in the mouse (13). In contrast, prolactin stimulates natural killer cell activity or lymphocyte proliferation in the mouse (26). However, these lymphocyte functions are enhanced in hamsters in short days, a photoperiod in which prolactin concentrations in circulation are typically reduced (42). For glucocorticoids, seasonal changes in circulation have not been studied in this hamster species but melatonin has been reported to ameliorate the immunosuppressive effects of glucocorticoid treatments (2). Whether photoperiod effects on immune cell function are mediated by a central action of melatonin remains open to study.

Melatonin may also act directly on immune cells themselves. Melatonin treatments are reported to enhance humoral and cell-mediated immunity (reviewed in Ref. 25 and 31). Melatonin has been found to activate natural killer cells, T helper 1 lymphocytes, and macrophages (1, 16). Moreover, specific melatonin binding sites on lymphoid tissues, lymphocytes, and macrophages have been identified (4, 24). Whether specific melatonin receptors are present on leukocytes or lymphoid tissue in seasonal rodents including the Siberian hamster remains to be determined. Support for the conclusion that melatonin may regulate immune cell function must take into consideration several caveats. Most studies have administered melatonin in doses and modes that are not physiological. Except for the important series of studies in the deer mouse (14, 30, 32), experimental models are typically inbred rodents for which the biological role for melatonin has not been defined. Thus the hypothesis that melatonin secretion from the pineal gland may mediate photoperiod effects on immune system functions remains to be tested.

In conclusion, the results suggest that certain functions by distinct lymphocyte populations are enhanced, whereas select activities by phagocytes are reduced in hamsters exposed to short compared with long days. The data raise the possibility that a repertoire of responses by the immune system to changes in day length may auger challenges presented by the environment during winter. Insufficient or inappropriate physiological responses to information provided by environmental cues may, in part, contribute to increases in disease and mortality. Because seasonal variability is evident in human immune function, the findings raise the possibility that photoperiod control of immune cell functions may be relevant to humans and other species that bear young throughout the year.

Perspectives