Tumoricidal Activity of Lymphokine-Activated Killer Cells During Acute Protein Restriction in the Cotton Rat (Sigmodon hispidus)

Comp. Biochem. Physiol. Vol. 119C, No. 2, pp. 149–155, 1998 Copyright  1998 Elsevier Science Inc. All rights reserved.

ISSN 0742-8413/97/$19.00 PII S0742-8413(97)00202-8

Tumoricidal Activity of Lymphokine-Activated Killer Cells During Acute Protein Restriction in the Cotton Rat (Sigmodon hispidus) R. L. Lochmiller, J. A. Sinclair, and D. P. Rafferty Department of Zoology, Oklahoma State University, Stillwater, Oklahoma, U.S.A. ABSTRACT. Habitat-induced alterations of immune system function have been implicated in the regulation of survival rates in wild herbivore populations. Protein availability in the diet has been shown to fluctuate with density and influence immunity in hispid cotton rats (Sigmodon hispidus), a common herbivorous rodent of the southeastern United States. In this study, we examined the impact of short-term, moderate restrictions in dietary protein on the tumoricidal activity of lymphokine-activated killer (LAK) cells in the spleen of subadult male cotton rats in captivity. Animals were fed complete, isocaloric diets containing either 20% casein (high quality diet), or one of three moderate levels of protein (10, 8, or 5% casein) for two weeks prior to assessing LAK cell activity in vitro in the presence of YAC-1 tumor cells. Moderate restrictions in protein resulted in depressed body growth, although all animals gained mass during the second week of the trial, without significant increases in food intake. Immune organ development and cellularity were suppressed in moderately restricted cotton rats when compared to those on a high quality diet. Tumoricidal activity of LAK cells against YAC-1 targets were significantly altered by diet treatments, being elevated in the group fed a diet containing 10% casein. There was a general tendency for increased LAK cell activity among those fed one of the three moderate quality diets, but observed suppressions in splenic cellularity tended to result in a slight decline in total lytic capacity of spleens. comp biochem physiol 119C;2:149–155, 1998.  1998 Elsevier Science Inc. KEY WORDS. Cotton rat, Sigmodon hispidus, immunity, natural killer cells, protein nutrition, nutritional ecology, malnutrition, LAK cells

INTRODUCTION Protein malnutrition has been shown to alter immune system function in vertebrates, however the nature of these alterations are variable, incorporating positive, negative, or normal responses (1,2,37). Variation in nutritional-induced immune responses of an animal depends upon their age at onset of nutrient restriction, reproductive status, severity of nutrient restriction, and probably species. Although few studies have explored how diet can potentially influence immunity and survival in wild animal species, responses are often very much different from those seen with standard laboratory animal models (14). Hispid cotton rats (Sigmodon hispidus; Rodentia: Muridae) have been used in several recent studies exploring how nutrition influences immunocompetence of wild smallmammal species. Cotton rats are generalist herbivores that occur in grass- or brush-dominated environments throughout the southeastern United States, and are well known for Address reprint requests to: Robert L. Lochmiller, PhD, Department of Zoology, Oklahoma State University, Stillwater, OK 74078, USA. Tel. (405)-744-9672; E-mail: [email protected]. Received 3 July 1997; accepted 30 October 1997.

their periodic fluctuations in density both within and between years (7,8,23). Demographic fluctuations have been linked to changing nutritional conditions in their habitat (6,20,29). Consequentially, ecologists have been interested in what affects the nutritional environment has on development and maintenance of immune system function in small mammals and their populations (13,15,17). Similar to laboratory rodents, involution or suppression of immune organ development is a common feature of dietary protein restriction in cotton rats, even at moderate levels of restriction (5,34). Elevated delayed-type hypersensitivity (DTH) responses and reduced hemolytic complement activity have been documented in weanling cotton rats subjected to severe protein restriction (34). Additionally, lymphoproliferative responses of mitogen-stimulated spleen cells from cotton rats on moderately restricted protein-calorie diets are normal or elevated, but the reverse has been observed for animals on severely restricted protein-calorie diets (16). Davis et al. (5) observed that protein-induced immune alterations in cotton rats may be the result of numerical changes in specific T- and B-lymphocyte subpopulations. This observation led us to explore the consequences of short-term nutritional restrictions of protein on specific

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lymphocyte populations such as those involved in innate, MHC-unrestricted cytotoxicity by lymphokine-activated killer (LAK) cells. Activity of LAK cells, a heterogenous population of natural killer cells and cytotoxic T-lymphocytes (4), are thought to be fundamental in regulating tumors, viral infected cells, and possibly bacterial and fungal infections in the host (10,32,36). Our primary objective in this study was to examine if moderate levels of protein in the diet of subadult male cotton rats adversely influences apoptotic activity of LAK cells against tumor target cells. MATERIALS AND METHODS Experimental Animals Forty 60–90 day-old male cotton rats averaging 66.5 6 5.7 g (SE) body mass were obtained from an outbred laboratory colony maintained by Laboratory Animal Resources, Oklahoma State University. Animals were housed in an approved, windowless animal-care facility under a 16L:8D illumination cycle (0600 to 2200 cst light) provided by fluorescent lighting at 23–24°C, 30–70% relative humidity, and 15 fresh-air changes/hr. Animals had access ad libitum to laboratory rodent chow (Purina 5001, St Louis, Missouri) and to clean tap water at all times prior to the start of experimental trials. All experimental procedures were approved in advance by our local Animal Care and Use Committee, Oklahoma State University, Stillwater. Experimental Design Cotton rats were weighed and paired in polypropylene cages (48 3 27 3 20 cm) with stainless-steel lids and randomly assigned to one of four experimental diets (10 per diet group) that varied in crude protein content. The control diet was a complete 20% casein-based ration (4.29 Kcal/g) formulated by PMI Feeds, Inc. (Richmond, Indiana). Adult cotton rats require a minimum of 12% crude protein in the diet to support reproduction (9) and probably 8% protein for maintenance in a non-reproductive animal (22). Three moderate-protein (10, 8, and 5% casein) diets were formulated by replacement of the casein source of protein in the control diet with an equal weight of sucrose to derive diets with a proximate analysis that was similar to the complete control diet, except casein. Each diet contained 10% cottonseed oil, 4% USP XIV Salt Mix (21415), and 2.1% USB Total Vitamin Supplement (23431) (United States Biochemical Co., Cleveland, Ohio). Diets were fed ad libitum in a pelleted form in ceramic crucibles for a period of two weeks; food intake was monitored by weighing food provided and remaining at 7-day intervals. At the end of the two week feeding trial, animals were anesthetized with methoxyflurane (Metofane; Pitman Moore, Mundelein, Illinois) vapors and final body mass recorded. Animals were euthanized by cervical dislocation

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and selected immune organs removed, trimmed of fat tissue, blotted dry, and weighed. Both absolute and relative (mg/ g body mass) organ size were determined. Lymphokine-activated Killer Cell Activity The spleen was removed aseptically, placed in preweighed 15 3 60 mm sterile petri dishes containing sterile RPMIS, and weighed to the nearest 0.1 mg. The RPMI-S was prepared by supplementing 100 ml of Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma Chem. Co., St Louis, Missouri) with 1.025 ml L-glutamine (Sigma, G7513, 200 mM solution), 1.0 ml Na pyruvate (Sigma, S8636, 100 mM solution), 1.0 ml non-essential amino acids (Sigma, M-7145, 100 3 solution), 1.0 ml penicillin-streptomycin (Sigma, P-0781, penicillin 10,000 U/ml and streptomycin 10 mg/ml), 100 µl 2-mercaptoethanol (Sigma, M7522, 50 µM), and 11.5 ml horse serum (Sigma, H-1263). Spleens were gently disrupted using a glass-on-glass tissue homogenizer containing 5 ml RPMI-S. The resulting cell suspension was decanted into sterile tissue culture tubes and centrifuged at 800 3 g for 8 minutes at 10°C. The supernatant was decanted and the cell pellet was resuspended in 10 ml tris-buffered ammonium chloride (0.83%, pH 7.2) to lyse erythrocytes. Following an additional three washes in RPMI-S, mononuclear cells were enumerated on an automated cell counter (System 9000, Serono Baker Instruments, Allentown, Pennsylvania). Viability using trypan blue was in excess of 95%. Splenocyte suspensions were adjusted to 5.56 3 106 cells/ml in RPMI-S. Splenic cellularity was calculated as the total splenocyte count and relative splenic cellularity (splenocytes/mg spleen). The tumoricidal activity of lymphokine-activated killer (LAK) cells was assessed by measuring cell death resulting from DNA fragmentation (apoptosis) using the Jabowakian apoptotic multifaceted assay [ JAM test; (18)]. Activated effector cells (LAK) were prepared by seeding 5.56 3 106 splenocytes in 2 ml RPMI-S into sterile 24-well tissue culture plates and culturing with 200 U of recombinant human interleukin-2 (No. 1147528, Boehringer Mannheim, Indianapolis, Indiana) for 18 hr at 37°C and 5% CO2. After incubation, LAK cells were washed once in RPMI-S, enumerated, and adjusted to 5 3 106 /ml in RPMI-S. Target YAC-1 tumor cells in log-phase growth were labelled with 3H-thymidine (10 µCi/ml) 24 hr prior to their use in the assay. Labelled target cells were washed three times in RPMI-S and numbers adjusted to 50,000 YAC-1/ ml. Apoptosis was measured at an effector:target cell ratio of 100:1 (100 µl volume each) in 96-well V-bottom tissue culture plates. Cells were mixed, plates centrifuged for 1 min at 50 3 g to pack cells, and plates incubated at 37°C with 5% CO2 for 4 hr. Eight negative control wells were run on each plate, consisting of labelled target cells only, to control for spontaneous DNA fragmentation. Cells were

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harvested on glass-fiber filter strips using a PHD Cell Harvester (Cambridge Technology, Watertown, Massachusetts) and vials counted (dpm) on a liquid scintillation counter. Percent specific killing was calculated by the formula: % Specific killing 5

S2E 3 100 S

where E is the experimentally retained DNA in the presence of LAK cells (in dpm) and S is the retained DNA in the negative control wells (in dpm). Total lytic capacity in the spleen was extrapolated by dividing total splenocyte yield by the number of lytic units (number of IL-2-activated splenocytes lysing 50% of targets at 100:1 effector:target ratio) in each culture (11). Statistical Analysis Data were tested for homogeneity of variances among treatment groups using a Levene’s test (31) and a test of normality (Proc Univariate; 30). All variables failing to meet the above assumptions were log-transformed prior to further statistical analysis. Differences among casein-diet groups were examined by analysis of variance [ANOVA; Proc GLM; (30)] with diet as a main factor, blocked by maternal dam and cage grouping. We further examined specific contrasts between the high quality (20%) and moderate quality (10, 8, 5% casein groups combined) diet groups using ANOVA. Organ weights were also tested between dietary groups using analysis of co-variance with body weight at time of termination as a covariate [Proc GLM; (30)]. RESULTS Initial body weights of subadult males were similar (P . 0.05) among diet groups, averaging 66.5 6 5.7 (SE) g (Table 1). Total and relative (g intake/g body mass) food intake during weeks 1 and 2 were similar (P . 0.05) among diet groups. Feed efficiency (g body mass change/g food intake) was negative for all moderate quality diet groups during week 1 of restriction, but was positive during the second week of restriction. Feed efficiency of the moderate quality diet groups was significantly lower than those on the high quality diet during both weeks 1 (P , 0.0001) and 2 (P 5 0.0009). Animals on the high quality diet had feed efficiencies that were about 2-fold greater than those on the moderate quality diets, which resulted in significantly (P , 0.0001) smaller sized animals in the moderate quality groups (72.9 6 2.9 g) compared to those fed a high quality diet (101.0 6 5.9 g) at the end of the trial. Final body mass was similar among animals fed the three moderate quality diets (Table 1). Cotton rats fed one of the three moderate quality diets showed significant reductions in the mass of the spleen

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(P 5 0.003) and thymus gland (P 5 0.028) compared to those fed a high quality diet, however, relative spleen mass (mg/g body mass) was not different (Fig. 1). Reductions in development of the spleen were most prominent in those fed the 8% casein diet, although differences among the three moderate quality diet groups were similar. Overall, mass of the spleen was 38% greater in the high quality group compared to those fed one of the moderate quality diets. Total splenocyte yields reflected the suppressed development of the spleen, with 90% greater yields among those fed the high quality diet compared to those on the moderate quality diets (P 5 0.012; Fig. 1). There was no difference in total splenocyte yield among the groups fed a moderate quality diet. Relative splenocyte yields (cells/mg spleen) tended to be lower in animals fed moderate compared to high quality diet, but differences were not significant (P 5 0.075). Mass of the popliteal lymph node was highly variable within groups fed either 8 or 10% casein diets compared to the high quality group, as revealed by a significant Levene’s test for homogeneity of variances (P 5 0.006); although, differences among means were not significant. Tumoricidal activity of LAK cells cultured with YAC-1 target cells differed significantly (P 5 0.05) among diet groups, with elevated killing activity by cells from animals fed the 10% casein diet when compared to those fed the high quality or 5% casein diets (Fig. 2). Killing activity was slightly elevated in the 8% casein group as well. Contrasts between those fed the high quality diet (65.4 6 3.0% lytic activity) and those fed one of the three moderate quality diets (72.0 6 2.3% lytic activity) showed a slight, overall tendency for greater tumoricidal activity of LAK cells with protein restriction (P 5 0.06). Despite the greater tumoricidal activity, the concommitant decrease in splenocyte yields with protein restriction resulted in a general tendency (P 5 0.066) for overall reductions in total splenic lytic activity among the moderate quality-fed groups (77.6 6 7.2 lytic units) compared to those on the high quality diet (122.4 6 18.3 lytic units; Fig. 2). DISCUSSION Lymphokine-activated killer cells consist of at least two main cell-types, namely, natural killer cells and a subpopulation of T-cytotoxic killer cells which display potent antitumor cytotoxicity (4). These cells appear as large granular lymphocytes and possess antigen-nonspecific, MHC-unrestricted cytotoxicity and thus are an important first-line of defence in the host’s immune system. Basal natural killer cell function is often low, but can be upregulated through IL-2 or IL-4 induction in vitro resulting in the heterogenous population of LAK cells (24,33). In preliminary experiments our laboratory has observed that basal natural killer cell tumoricidal activity against YAC-1 targets is extremely low in the cotton rat (,10% specific lysis at a 100:1 ef-

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TABLE 1. Body mass, food intake, and feed efficiency of sub-adult male cotton rats (Sigmodon hispidus) fed varying levels

of protein as casein in their diet for two weeks. Significant differences (P , 0.05) among diets are depicted by different superscripts. Casein content of diet 5%

Parameter Initial body mass (g) Final body mass (g) Food intake g/g body mass/day) Week 1 Week 2 Feed efficiency (g body mass change/g good) Week 1* Week 2

X 66.6 74.8 a

8% SE 4.4 4.0

X 66.3 65.0 a

10% SE 5.6 5.1

X 65.6 79.2 a

20% SE

X

SE

6.3 5.5

67.5 101.1 b

6.8 6.0

0.164 0.143

0.028 0.023

0.112 0.133

0.022 0.027

0.161 0.136

0.028 0.024

0.110 0.091

0.006 0.005

20.11 a 0.094 a

0.014 0.011

24.288 a 0.078 a

2.814 0.053

20.132 a 0.151 ab

0.132 0.019

0.277 b 0.266 b

0.036 0.021

*The large SE for 8% casein group was due to two individuals that lost a disproportionate amount of body mass.

fector: target ratio) compared to laboratory Mus. However, killer cells of cotton rats were found to be easily upregulated in vitro, with peak LAK activity observed after 18 hr of IL2 stimulation in culture (19). Overall percent specific lysis of LAK cells against YAC-1 tumor cells in a 4-hr culture with a 100 :1 effector: target ratio was about 65% in the cotton rat, which was comparable to levels reported for various laboratory Mus strains (25). Sensitivity of cytotoxic lymphocytes to the nutritional environment of laboratory rodents is highly variable and appears to depend upon age of experimental subjects, diet or type of malnutrition, and level of activation of killer cells. Reynolds et al. (25) observed that sub-adult CBA/J mice fed a 2.5% protein diet for two weeks possessed normal basal natural killer cell and IL-2-stimulated LAK activity, but showed depressed natural killer cell function when activated in vivo with poly I:C (polyinosinic:polycytidylic acid). These observations were in agreement with those of Saxena et al. (28) who observed that six-week old sub-adult C57B1/ 6 female mice fed low protein diets (4%) for two weeks show no adverse effect on basal natural killer cell or IL2-stimulated LAK cell tumoricidal activity against YAC-1 cells. In comparison, weanling C57BL/6J and CBA/J mice fed a very low protein diet (0.5% protein) for two weeks showed depressed natural killer cell activity against YAC1 target cells compared to those fed a 19% protein control diet (11,12). Thus, killer cell activity of sub-adult cotton rats in our study appeared somewhat more sensitive than laboratory mice at a more moderate level of protein restriction (i.e., 10% casein). However, the apparent elevated activity that we observed at this level protein restriction differed from the normal to depressed responses reported in previous studies with laboratory mice. It is also important to note that the positive gains in body mass that we observed in sub-adult cotton rats fed the moderate quality

diets in week 2 suggested that these animals were in positive nitrogen-energy balance. Innate immune responses of animals appear to respond to dietary restriction or starvation differently than when subjected to low protein diets consumed ad libitum. In fact, long-term dietary restriction (.3 months) may actually improve natural killer cell responses in older mice (35). Hybrid mice restricted in their intake of energy (without malnutrition) from weaning to 3 months-of-age show depressed basal natural killer cell activity, but normal to slightly greater Poly I:C-activated killer cell responses compared to unrestricted controls (35). Similar reductions in natural killer cell activity to YAC-1 cells have been reported in young CBA mice subjected to 3 to 6 days of acute starvation (27). These types of responses of laboratory rodents are similar to the reduced innate killer cell responses of peripheral blood lymphocytes of children experiencing combined proteinenergy malnutrition (26). The above literature shows the sensitivity of young, developing immune systems relative to those of older adults when confronted with nutritional stressors. Although basal natural killer cell activity is often unaffected by proteinrestricted diets in some rodent strains, reductions in the total population of killer cells may compromise overall immune surveillance responsibilities of LAK cells. Involution of the spleen during protein malnutrition is a common response of laboratory rodents, which is characterized by a much reduced population of mononuclear immune cells (11). Thus, the total units of lytic activity available from innate killer cells in the spleen can be much reduced during protein malnutrition, despite near normal levels of activity for individual cells (28). Splenic involution with corresponding declines in cellularity have been well documented in weanling to adult cotton rats subjected to 4% crude protein diets (21,34). However, cotton rats that are restricted

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FIG. 1. (A–F) Mass and cellularity of selected immune organs of sub-adult male cotton rats (Sigmodon hispidus) fed diets containing 5, 8, 10, or 20% casein for two weeks. Values are reported as means (6SE bar) for n 5 10; means with different letters above bars are significantly different (P , 0.05) from one another. Results of specific contrast comparisons between those fed high (20%) and moderate (10, 8, and 5% casein diets pooled) quality diets are shown as a P-value at the topright of each graph.

in their intake of a complete diet at moderate to severe (40% ad libitum intake) levels do not show declines in splenic cellularity or mass (16). The sub-adult cotton rats in this study showed moderate reductions in splenocyte yields compared to controls, even when fed as high as 10% casein in their diet for two weeks. The tendency towards an elevation in tumoricidal activity of LAK cells was not sufficient to offset the moderate decline in splenocyte yields, resulting in a general tendency of reduced total lytic capacity in the spleen. These observations suggest that more severe (,5% protein) or prolonged restrictions (.2 weeks) of dietary protein may have exacerbated these immune alterations in sub-adult males. Changes in killer cell activity in response to dietary pro-

tein-calorie malnutrition are probably not attributable to alterations in numbers of natural killers cells in the spleen. Use of murine NK-specific surface markers (NK 1.1) revealed no proportional changes in their representation in the spleen of mice during severe protein-calorie malnutrition (12); reductions in splenic natural killer cells have been documented during vitamin A deficiencies in rats (3). The generation of LAK cells in vitro was not greatly altered by two weeks of protein malnutrition in the cotton rat, suggesting that natural killer cells are capable of responding to cytokine messengers for upregulating or expanding their populations, at least in sub-adults. These results indicate that the innate killer cell population in the spleen of cotton rats can be altered by moderate

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simultaneously exposed to multiple environmental stressors. Given that the magnitude of the alterations in LAK cells we observed in cotton rats was not great, it is possible that survival would not be compromised as a result of these lesions during moderate protein restriction. The authors greatly appreciate the assistance of Lee Jones in the laboratory and Dr. M. E. Payton, Oklahoma State University, Department of Statistics, with data analysis. Financial support for this research was provided through the National Science Foundation (IBN-9318066) and the Department of Zoology, Oklahoma State University. This research was approved by the Oklahoma State University Institutional Animal Care and Use Committee as protocol number 236.

References

FIG. 2. (A, B) Tumoricidal activity (% apoptosis) against YAC-1 cells by lymphokine-activated killer (LAK) cells of subadult male cotton rats (Sigmodon hispidus) fed diets containing 5, 8, 10, or 20% casein for two weeks. Total lytic capacity is a relative measure of how much tumoricidal activity exists in the spleen and is extrapolated from both total splenic cellularity and amount of LAK cell activity. Values are reported as means (6SE bar) for n 5 10 (9 in 8% group); means with different letters above bars are significantly different (P , 0.05) from one another. Results of specific contrast comparisons between those fed high (20%) and moderate (10, 8, and 5% casein diets pooled) quality diets are shown as a P-value at the top-right of each graph.

restrictions in dietary protein, such as those levels used in our experimental design. Additionally, this supports the hypothesis that LAK cell immune responses are more sensitive in wild cotton rats than many laboratory Mus strains. However, the consequences in terms of disease resistance from elevated tumoricidal activity or a potential decline of total lytic capacity in the spleen is not known, especially under natural conditions of the wild where cotton rats are often

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