An in vivo and in vitro study of selenium deficiency and infection in rats

J. COMP.

1986

PATH.

AN

IN

VOL.

96

VIVO AND DEFICIENCY

IN

VITRO STUDY AND INFECTION

OF SELENIUM IN RATS

BY

R. BOYNE,

J. R. ARTHUR and A. B. WILSON

Row&t Research Institute, Bucksburn,

Aberdeen. AB2 9SB. Scotland, U.K.

INTRODUCTION

The micronutrient selenium (Se) plays an important role in the humoral and cellular immune systems of animals (Spallholz, 1981). In in vitro tests of function, Se deficiency impairs the ability of phagocytic neutrophils from rats (Serfass and Ganther, 1975), cattle (Boyne and Arthur, 1979, 1981) and mice (Boyne and Arthur, 1985) to kill ingested Candida albicans organisms. Several metabolic defects could contribute to this impaired activity. Neutrophils from Se-deficient goats exhibit reduced migration and chemiluminescence compared with neutrophils from Se adequate animals (Aziz, Klesius and Frandsen, 1984). In bovine neutrophils, Se deficiency impairs glucose oxidation (Arthur and Boyne, 1985) and production of hydroxyl radicals which are used in the microbicidal process (Arthur, Boyne, Hill and Okolow-Zubkowska, 1981). In rat neutrophils, microtubules are disrupted in Se deficiency, a defect also thought to contribute to impaired candidacidal activity (McAllister, Harris, Baehner and Boxer, 1980). Selenium deficiency adversely affects the humoral immune system of the body, which may also be stimulated by Se intakes in excess of normal dietary requirements (Spallholz, 1981). Probably as a result of defects in phagocytic and humoral immune function, Se deficiency increases the susceptibility of mice to Diplococcuspneumoniueand C. ulbicunsinfections (Spallholz, 198 1; Boyne and Arthur, 1985). Pigs on a diet supplemented with Se (0.2 mg per kg diet) have an increased resistance to infection by Treponemuhyodysenteriaecompared with Se-deficient control animals (Tiege, Tollersund, Lund and Larsen, 1982). In contrast, Se deficiency decreases the severity of Salmonella &phimurium infection of rats (Boyne, Mann and Arthur, 1984) and Plasmodium berghei, Listeria monocytogenes and Pseudorabies virus infections in mice (Murray and Murray, 1985). To further clarify the relationships between Se deficiency, phagocytic activity and resistance to infection, we have investigated the ability of severely Se-deficient rats to survive Staphylococcusaureusinfections and, with bacterial and or yeast cells, have compared the phagocytic and microbicidal activities of neutrophils and macrophages elicited from the peritoneal cavity of Se-depleted and supplemented rats.

0021-9975/86/040379+08

$03.00/O

0

1986

Academic

Press

Inc.

(London)

Limited

380

R. MATERIALS

Experimental

BOYNE AND

et

al.

METHODS

Animals

Male, specific pathogen-free, Hooded Lister rats of the Rowett Institute strain were used in all experiments. Animals consumed Se-deficient (0.01 mg Se per kg) or supplemented (0.1 mg Se per kg) diets for 8 weeks from weaning before they were used in experiments. Selenium deficiency did not adversely affect growth of the rats, which weighed approximately 300 g. The development of Se deficiency was followed by monitoring changes in whole blood glutathione peroxidase activity (Boyne etal., 1984). Phagoqtic

and Microbicida

Activity of Neutrophils

and Macrophages

Phagocytic cells were obtained from rats by peritoneal lavage with sterile saline, 4 to 18 h (neutrophils) or 7 days (macrophages) after an intraperitoneal (IP) injection of 40 ml of 0.05 per cent glycogen in physiological saline. In vitro studies of phagocytic and microbicidal activity with C. albicans were carried out by the methods of Boyne and Arthur ( 198 1) and with S. typhimurium and S. aureus by the methods of Van Furth, Van Zwet and Leijh (1978). Nitroblue tetrazolium (NBT) reduction by resting and endotoxin-stimulated cells was determined by the methods of Boyne and Arthur (1981). In vivo Studies Experiment I consisted of three trials, in each of which 7 Se-depleted and 7 Sesupplemented rats were injected IP with 8.0 X lo7 5’. aureus organisms in 1 ml saline and survival of rats was studied for a period of 15 days. Experiment 2 consisted of 4 trials, in which 30 Se-depleted and 28 Se-supplemented rats were injected IP with 4.0 X 10’ S. aureusorganisms in 1 ml saline and rat survival studied for 15 days. All rats had been injected IP, 18 h previously, with 40 ml of 0.05 per cent glycogen in physiological saline to stimulate production of intraperitoneal neutrophils, producing an organism-to-neutrophil ratio of approximately 5 to 1. Experiment 3 consisted of three trials, in each of which 4 Se-depleted and 4 Sesupplemented rats, preinjected with glycogen-saline, were injected IP with 5.0 x 10’s. aureus organisms in 1 ml saline. At 30 min, 1, 2 and 4 h after 5’. aureus injection, 1 Sedepleted rat and 1 Se-supplemented rat was killed and cells ( > 95 per cent neutrophils) washed from the peritoneal cavity with ice-cold sterile saline (final volume 20 ml). Viable intracellular and extracellular S. aureus organisms were determined in each neutrophil suspension using the techniques of Van Furth et al. (1978). Neutrophils in each sample were counted in a haemocytometer. Statistical Analysis Results of rat survival experiments were analysed by a non-parametric test (MannWhitney U test). Other comparisons between Se-deficient and Se-supplemented groups were by Student’s t test.

RESULTS

In vitro Studies There was no difference between the neutrophils of Se-depleted and Sesupplemented rats in their ability to phagocytose C. albicans; however, candidatidal activity differed significantly (Table 1). NBT reduction was similar in resting Se-depleted and Se-supplemented neutrophils. However, following

INFEcTIoNs

IN SE-DEFICIENT

RATS

381

endotoxin stimulation, the increase in NBT reduction in the Se-supplemented neutrophils was significantly greater than in Se-depleted neutrophils (Table 1). Neutrophils from Se-depleted and Se-supplemented rats did not differ in their ability to phagocytose and kill S. aureus or S. ~phimuriu’nz organisms (Table 2). Both the reduction in ability to phagocytose and kill C. albicans and the activities of glutathione peroxidase were reduced significantly in macrophages from Se-depleted rats (Table 3).

TABLE

1

COMPARISON OF PHAGOCYTIC, CANDIDACIDAL NEUTROPHILS FROM SE-DEPLETED (-SE)

AND NBT REDUCTION AND SE-SUPPLEMENTED

Phagocytic acti& - se Test

Candidacidal + se

activity

-se

+.Ye

organism

C. albicans

226f3

22a*2

reduction

53*

IOfl

Resting neutrophils

NBT

ACTIWTIES OF (+SE) RATS

I*

Stimulated neutrophils

- se

+ se

a*1

IOfl

- Se

+Se

18f2

46f2*

Results of 6 tests expressed as M~XIS+S.E.M. *P
TABLE COMPARISON

2

OF PHAGOCYTIC AND BACTERKXDAL ACTIVITIES OF NEUTROPHILS SE-DEPLETED (-SE) AND SE-SUPPLEMENTED (+SE) RATS

Phagocytic activity - se Test

-Sl?

activity +se

organism

S. typhimurium Test

+Se

Bactericidal

FROM

40.4

f 3.7

40.2 f 4.6*

9.07 f 0.30

9~4lf0~51t

organism

S. aurew

75.6 f 3.8

72.6f4.8t

1 I-07f5.59

11.75f0.777

Results expressed as Means* S.E.M. of * 8 tests: t 3 tests. Phagocytic activity is expressed as the percentage of available organisms (original organism to neutrophil ratio 1 to 1) phagocytosed in 30 min. Bactericidal activity is expressed as the percentage of phagocytosed organisms still viable at 30 min.

382

R.

TABLE COMPARISON

et al.

BOYNE 3

OF PHAGOCYTIC AND CANDIDACIDAL ACTWITIES AND SE-SUPPLEMENTED RAT MACROPHAGES

Se-depleted macrophages (7) Phagocytic Range Candidacidal Glutathione activity

activity activity peroxidase units per mg protein

473f7 441 to 496 17fl 0.0834f

0.02

OF SE-DEPLETED

Se-supplemented macrophages (8) 501*5* 473 to 518 55*1t 1.514+0.118+

Results expressed as Means f S.E.M. Number oi tests in parenthesis. *p
organisms macro-

In vivo Studies

Experiment 1. There was no difference in percentage survival (81 and 86 per cent) between the Se-depleted and Se-supplemented rats after injection of 8 X 10’ S. aureus organisms. Fifty per cent of survivors in each group had abscesseswithin the peritoneal cavity or scrotum, accompanied by enlarged but apparently normal spleens, group mean weights of which were 0.473 f 0.062 per cent of body weight (BW) ( f S.E.M.) in the Se-depleted and 0.691 f O-153 per cent of BW in the Se-supplemented survivors. The difference in weight was not statistically significant (P> O-10). Spleen mean weights were 0.20 1 f 0.006 per cent BW in the Se-depleted and 0.198 f 0.005 per cent BW in Se-supplemented survivors without visible lesions. Experiment 2. In contrast to the previous experiment, the death rate of Sedepleted rats was significantly greater (PC 0.05) than that of Se-supplemented rats during days 1 to 8 after S. aureus injection, although by day 12 the difference just failed to reach significance (P>O.O5<0*1) (Fig. 1). Abscesses accompanied by splenic enlargement were present in 50 per cent of the survivors of both groups. Group mean weights of spleens were 0.854 f 0.137 per cent of BW in Se-depleted and l-071 f O-134 per cent of BW in Sesupplemented animals. In those survivors without visible abscessesin the peritoneal cavity or scrotum, spleens were 0.2 19 f 0.016 per cent and 0~232f0~008 per cent of BW in Se-depleted and Se-supplemented animals, respectively. Neither difference between Se-depleted or Se-supplemented groups was statistically significant. Experiment 3. There were no differences between Se-depleted and Sesupplemented rats, either in the number of peritoneal neutrophils induced by injection of glycogen, or in the ability of these cells to phagocytose and kill ingested S. aweus organisms (not shown). The mean numbers of viable organisms per peritoneal cavity 30 min after injection and for the remaining 3.5 h, expressed as per cent of inoculum, was 2.29 f 0.61 per cent and 2.00 f 0.63 per cent for Se-depleted and Se-supplemented animals, respectively (Mean f S.E.M., 12 tests per group).

INFECTIONS

I 2

IN

I 4

SE-DEFICIENT

I 6

383

RATS

I 8

I IO

I 12

I

Days Fig.

1. Cumulative injection Experiment

mortality of S. aweus. 2.

of Se-depleted Animals and

rats (O-O) pretreatments

and Se-supplemented rats (O-O) following IP are described in Materials and Methods; in viva

DISCUSSION

The markedly impaired candidacidal but unimpaired phagocytic activities of neutrophils from Se-deficient rats (Table 1) are consistent with observations previously reported for neutrophils from Se-deficient rats, mice and cattle (Boyne and Arthur, 1979, 1981, 1985; Serfass and Ganther, 1975). Consistent with impaired function was the inability of stimulated Se-deficient neutrophiis to reduce NBT (Table l), indicative of decreased generation of O,- derived radicals essential for microbicidal activity (Boyne and Arthur, 1981). Decreased seleno-enzyme glutathione peroxidase activity is thought to be the cause of depressed microbicidal activity (Serfass and Ganther, 1976) and O,derived radical production in the Se-deficient neutrophil (Arthur et al., 1981; Arthur and Boyne, 1985). The 95 per cent decrease in glutathione peroxidase activity (Table 3) may similarly be responsible for the impaired in vitro candidacidal activity observed with Se-deficient macrophages. The small 5 per cent change in macrophage phagocytosis of C. albicans, although statistically significant, is unlikely to have any biological significance (Table 3). Although defects in neutrophil candidacidal activity have been associated with the increased susceptibility to C. albicans infections of Se-deficient mice (Boyne and Arthur, 1985), it now appears possible that similar defects in macrophage function could also have contributed to this effect. Despite effects on neutrophil candidacidal activity, Se deficiency did not affect killing of S. aureus or S. pphimurium organisms by neutrophils. Although overall phagocytosis of either organism was not affected by Se deficiency, S. aureus was more efficiently ingested than 5’. typhimurium by neutrophils from both Se-deficient and Se-supplemented rats in the tests (Table 2). Chatelet, Burk: Shirley and Szejda (1977) were unable to demonstrate any &ect sf Se defici.ency on the ability of rat neutrophils to kill ingested cells oi

384

R.

BOYNE

et

al.

Proteus mirabilis. Thus defects in neutrophils due to Se deficiency will not always result in impaired function. Killing of Candida organisms may put greater

oxidative and metabolic demands on the neutrophil than killing of the much smaller bacteria and thus overwhelm the compromised antioxidant system of the Se-deficient neutrophil. The average volume of a C. albicans cell is 30 to 50 times that of a S. &phimurium or S. aureus organism and Candida to neutrophil ratios were double the bacteria to neutrophil ratios in the tests of function. Additionally, killing of bacteria by neutrophils could involve lytic processesnot affected by Se deficiency. The effects of Se deficiency on neutrophil and probably macrophage function can explain the susceptibility of Se-deficient mice to a systemic C. -1bicans infection (Boyne and Arthur, 1985). Similarly, the lack of effect of Se deficiency on the response of rats to the lower dose of S. aureus (in vivo, Exp. 1) is consistent with the unimpaired ability of neutrophils to kill S. aureus. However, after stimulation of neutrophil migration to the peritoneal cavity, an IP injection of S. aureus caused a significantly greater death rate in Se-deficient than in normal rats. The glycogen preinjection was used to allow interaction between the bacteria and neutrophils before the micro-organisms encountered any other of the microbicidal systems of the body. Despite the initial significantly differing death rates of the glycogen pretreated Se-deficient and Se-supplemented rats, there was no difference between the groups in total numbers of peritoneal neutrophils and clearance and survival of S. aureus. The differing death rates are therefore unlikely to be due to effects of Se deficiency on neutrophil function. The higher dose of 6’. aureus (4.0 X 10’ organisms) probably overwhelmed other defective humoral or cellular microbicidal systems of the Se-deficient animals. Spallholz, Martin, Gerlach and Heinzerling ( 1973) have demonstrated that spleen plaque-forming cells were decreased and production of antibodies was impaired in Se-deficient mice. These effects, in addition to those on neutrophil function, would adversely affect the response of animals to infection. Gyang, Stevens, Olson, Tsitsamis and Usenik (1984) have shown that injection of Se-deficient cattle with Se and vitamin E enhances neutrophil ability to kill 5’. aureus. Since Se and vitamin E were not administered separately, the effects on neutrophil activity cannot be attributed to Se alone. The trend to larger spleen size in Se-supplemented rats with residual peritoneal or scrotal abscesses (in vivo Expts 1 and 2) suggests that Se deficiency could impair the response of the reticula-endothelial system (RES) to infection. The lack of effect of Se deficiency on spleen size in rats showing no lesions further implies that demonstration of effects on RES function may depend on an infective stress in the animal. Despite adverse effects on antimicrobial systems, Se deficiency will protect rats against S. typhimurium infection (Boyne et al., 1984) and mice against infections of P. berghei, L. monocytogenes and Pseudorabies virus (Murray and Murray, 1985). Some of these organisms cause intracellular infections, hence phagocytic cell activity in body fluids may not be of major importance in resistance to chronic infections. Possibly of greater significance in the Sedeficient host is growth of the infective micro-organisms, which may be

INFECTIONS

IN

SE-DEFICIENT

RATS

385

inhibited by lack of Se or changes in the metabolism of the host cell. This type of effect occurs in iron deficiency, which can protect against infections by decreasing free iron in the tissues of deficient animals and thus inhibiting growth of infective organisms (Weinberg, 1984). The effects of Se deficiency on host responses to infection depend on complex interactions between (a) defects in the many microbicidal systems of the body, (b) the ability of micro-organisms to grow in the deficient animals and (c) the possibility, not discussed here, that the ability of micro-organisms to penetrate the tissue of the host may be affected. If other relevant interacting effects of Se deficiency are taken into consideration, in vitro tests of neutrophil microbicidal activity will be useful in predicting the response of Se-deficient animals to infection. SUMMARY

Selenium deficiency in rats impairs the ability of neutrophils and peritoneal macrophages to kill Candida a&cans organisms in vitro. In contrast, killing of Salmonella Qphimurium and Stafihylococcus aweus organisms is unaffected by the deficiency. Survival of rats after intraperitoneal injection of 8 X IO7 S. aureus organisms was not affected by Se deficiency, but a 5-fold increase in the dose (4 X IO8 S. aureus organisms) led to a significantly greater mortality in the Sedeficient rats. ACKNOWLEDGMENTS

We are grateful to the staff of the Small Animal of the rats and to Mr I. McDonald for statistical

Unit at the Rowett Institute analysis of the results.

for care

REFERENCES

Arthur, J. R., Boyne, R., Hill, H. A. 0. and Okolow-Zubkowska, M. J. (1981). The production of oxygen-derived radicals by neutrophils from selenium-deficient cattle. FEBS Letters,135, 187-190. Arthur, J. R. and Boyne, R. (1985). S u p eroxide dismutase and glutathione peroxidase activities in neutrophils from selenium-deficient and copper-deficient cattle. Life Sciences, 36, 1569-1575. Aziz, E. S., Klesius, P. H. and Frandsen, J. C. (1984). Effects of selenium on polymorphonuclear leukocyte function in goats. AmericanJournal of Veterinary, Research,45, 17 15-l 7 18. Bass, D. A., De Chatelet, L. R., Burk, R. F., Shirley, P. and Szejda, P. (1977). Polymorphonuclear leukocyte bactericidal activity and oxidative metabolism during glutathione peroxidase deficiency. Infectionand Immunigv,18, 78-84. Boyne, R. and Arthur, J. R. (1979). Alterations in neutrophil function in selenium deficient cattle. Journal of Comparative Patholou, 89, 151-158. Boyne, R. and Arthur, J. R. (1981). Effects of selenium and copper deficiency on neutrophil function in cattle. Journal of Comparative Pathology,91, 271-276. Boyne, R. and Arthur, J. R. (1985). Effects of Candidaalbicansinfection on selenium deficient mice. TraceElements in Man andAnimals TEMA-5, C. F. Mills, I. Bremner and J. K. Chesters Eds, Commonwealth Agricultural Bureaux, Slough, U.K. pp. 240-243. Boyne, R., Mann, S. 0. and Arthur, J. R. (1985). The effect of Salmonellatyphimurium infection on selenium deficient rats. Microbios Letters,27, 83-87.

386

R.

BOYNE

et al.

Gyang, E. O., Stevens, J. B., Olson, W. G., Tsitsamis, S. D. and Usenik, E. A. (1984). Effects of selenium-vitamin E injection on bovine polymorphonucleated leukocytes phagocytosis and killing of Staphylococcus aureus. American Journal of Veterinary Research, 45, 175-I 77. McAllister, J., Harris, R. E., Baehner, R. L. and Boxer, L. A. (1980). Alteration of microtubule function in glutathione peroxidase-deficient polymorphonuclear leukocytes. Journal of the Reticuloendothelial Society, 27, 59-66. Murray, J. M. and Murray, A. B. (1985). The effect of selenium deficiency and repletion on host resistance to infection. Trace Elements in Man and Animals TEMA5, C. F. Mills, I. Bremner and J. K. Chesters Eds, Commonwealth Agricultural Bureaux, Slough, U.K. pp. 244-247. Serfass, R. E. and Ganther, H. E. (1975). Defective microbicidal activity in glutathione peroxidase deficient neutrophils of selenium deficient rats. Nature, 225, 640-64 1. Serfass, R. E. and Ganther, H. E. (1976). Effects of dietary selenium and tocopherol on glutathione peroxidase and superoxide dismutase activities in rat phagocytes. Life Sciences, 19, 1139- 1144. Spallholz, J. E. (1981). Selenium what role in immunity and immune cytotoxicity? In Selenium in Biology and Medicine, AVI Publishing Co., Westport Conn., U.S.A., pp. 103-l 17. Spallholz, J. E., Martin, J. L., Gerlach, M. L. and Heinzerling, R. H. (1973). Enhanced IgM and IgG titres in mice fed selenium. Infection and Immunity, 8, 841-842. Tiege, J., Tollersund, S., Lund, A. and Larsen, H. J. (1982). Swine dysentery: the influence of dietary vitamin E and selenium on the clinical and pathological effects of Treponema hyodysenteriae infection in pigs. Research in Veterinary Science, 32, 95-100. Van Furth, R., Van Zwet, T. J. L. and Leijh, P. C. (1978). In Handbook of Experimental Zmmunolou, Volume 2, 3rd Edit. D. M. Weir, Ed., Blackwell Scientific Publications, Oxford, pp. 32.1-32.19. Weinberg, E. D. (1984). Iron withholding: A defense against infection and neoplasia. Physiological Reviews, 64, 65- 102. [Received for publication,

April

9th, 19851