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Effect of dietary magnesium on Ascaris suum infections of mice
BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
42, 95-104 (1989)
Effect of Dietary Magnesium on Ascaris suum infections of Mice HAROLD
E.
LAUBACH
Department of Microbiology, Southeastern University of the Health Sciences, 1750 N. E. 168th Street, North Miami Beach, Florida 33162 Received January 26, 1989, and in revised form June 1, 1989
Magnesium deficiency in mammals is characterized in its earliest stage by a series of clinical and laboratory manifestations, such as a pronounced hyperemia of the skin, leukocytosis, and eosinophilia (1,2). These characteristics develop in 6 to 9 days and are suggestive of a transient hypersensitive state. If animals are sacrificed after 21 to 28 days on a magnesium deficient diet, many microscopic lesions are found in various tissues. The lesions are characterized by focal perivascular collections of inflammatory cells which may be associated with cell necrosis. The histological picture resembles that seen in delayed hypersensitivity reactions. A deficiency in dietary magnesium has been shown to have a marked effect on the immune response in mice (3). Mice on a magnesium-deficient diet were immunized with sheep red blood cells and the immune response was studied by estimating the number of spleen antibody-forming cells capable of lysing the red blood cells and by a cytoadhesion test to determine the number of rosette-forming cells, T lymphocytes. Magnesium deficiency produced a drastic fall in the primary and secondary immune responses, as measured by the number of spleen antibodyforming cells. The number of rosette-forming cells was also much lower in the spleens of magnesium-deficient mice. With these present data on magnesium deficiency, the question was posed as to its effect on the inflammatory response to helminth infections. Previous research has demonstrated that there are increased levels of lysophospholipase (LPL: EC 3.1.1 S) in the lungs of animals infected with Angiostrongyhs cantonensis (4,5), Nippostrongylus brusiliensis(6), and Strongyloides ratti (7). Increased LPL activity correlates with increased tissue eosinophilia and with the presence of the parasites in the tissues. An anamestic response was observed. As it has been shown that T-cell numbers are reduced when mice are placed on magnesium-deficient diets (3) and that T lymphocytes are involved in LPL activity (8), it would be interesting to study the synergistic action of magnesium deficiency on changes in the inflammatory response to infection with Ascaris suum in mice. A model was used that included A. suum infection as the inducer of inflammation 95 0885-4505189 $3.00 Copyright Q 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.
96
HAROLD
E. LAUBACH
and the effect of magnesium deficiency was assessed by measuring the numbers of eosinophils and larvae found in liver or lung tissues and the LPL activity associated with these infected organs. MATERIALS
AND METHODS
Male, weanling BALB/c AnNCr/BR Mice (Charles River Breeding Laboratories, Wilmington, MA) were divided into 20 groups of 10 mice each and fed defined diets. The mice were maintained at 25°C in a well-ventilated animal room. Mice were divided into four sets, each with five groups of 10 mice each. One group of 10 mice in each of the four sets of mice was dietary magnesium control mice. Control groups of mice received a nutritionally defined diet consisting of 16% casein, 4% lactalbumin, 4% cellulose, 4% of a complete vitamin mixture (9), 48% wheat starch, 16% sucrose, 4% corn oil, and a complete mineral oil mixture (699 g CaCO,, 966 g KH2P0,, 1220 g CaHOP,, 400 g MgS0,J7H20, 508 g NaCl, 191 g Na,Se03, 0.03 g KIO,, 17.3 g MnS04/H20, 2.2 g CuS04/4H20, 2.6 g ZnCo,, 1.2 g KZCr207, 0.003 g NaF, and 10.4 g FeS0,/7Hz0 per loo-kg diet). This defined diet was supplied in sufficient quantity so that the mice could eat unlimited quantities. Mice were also given demineralized water in unlimited quantities, The experimental magnesium diet groups of mice received the defined diet modified in a way to provide 0.0, 200.0, 600.0, or 800.0 g MgS04/7HZ0 per 100 kg diet. These mice were also given their diets and demineralized water in unlimited quantities. Viable eggs of female A. suum worms were digested out of the distal third of the uterus using NaOH, washed with buffered saline, and placed into petri dishes in dilute sulfuric acid. The eggs were allowed to embryonate and were then washed with buffered saline prior to use (10). Mice were inoculated, per OS, with 1000 A. suum infective eggs by means of a dosing needle inserted into the esophagus. Groups of mice were infected 21 days after they were placed on their various magnesium diets. Control mouse groups were infected in the same manner as the experimental mouse groups. The four sets of animals consisted of two sets of mice immunized with A. suum infective eggs and two sets of nonimmunized mice. The first set of mice were nonimmunized mice that were challenged with 1000 A. suum eggs. The second set were also nonimmunized mice but were not challenged with eggs. The third set were immunized mice and were challenged with 1000 A. suum eggs. The fourth set of mice were immunized but were not challenged with eggs. Immunized mice were infected with 1000 A. suum infective eggs, per OS, 30 days before challenge with 1000 A. suum eggs. Ali four sets of mice had five mice per group killed 2 days after challenge with eggs and the remaining five mice in each group were killed 7 days postinfection (pi). At necropsy, total body and blotted lung and liver weights were recorded. One portion of lung and liver tissue from each mouse was crushed with a tissue grinder and digested in a solution of 0.7% pepsin and 1.0% hydrochloric acid. The digest was searched for larvae, in small volumes in a petri dish, until all of
RESPONSE
TO MAGNESIUM
DEFICIENCY
97
the digest was examined. Total larvae present in the lungs and liver of each mouse were recorded. A second portion of liver and lung tissue from each mouse was fixed in 10% buffered formalin immediately after death. Tissue samples were stained with hematoxylin-eosin, embedded in paraffin, sectioned, and mounted onto slides. Stained sections were examined for pathological changes at 400X using brightfield illumination. Cell types in the area of A. suum larvae, but not adjacent to the larvae, were observed for each tissue preparation. A total of 10 fields was examined for each tissue sample and expressed as average counts per field at 400X illumination. A third portion of lung and liver tissue from each mouse was assayed for LPL activity according to the method of Ottolenghi (11). Lung and liver tissues were weighed, mixed I:20 with cold 12.5% glycerol solution, and ground up. Fivemicroliter samples of the mixtures were added to each of four test tubes and equilibrated in a 37°C bath for 4 min. Three-tenths milliliter of prewarmed lysophosphatidylcholine (substrate for LPL) solution (20 kl/ml) was added to each of the test tubes and incubated at 37°C for 30 min. Control tubes consisted of four 5~1 samples of the lung and liver mixtures without lysophosphatidylcholine solution added. The enzyme reactions were stopped by the addition of 0.1 ml of 2 NH2S04, 1.0 ml of isopropyl alcohol, and 0.4 ml of water. Two milliliters of heptane was added to each tube and mixed. The heptane was removed and titrated with 0.01 N NaOH and bromthymol blue while bubbling nitrogen into the test tube. LPL activity was expressed as micromoles per gram tissue/per hour. The average & standard deviation of the body weight and lung and liver weights, LPL levels, eosinophil numbers, and the numbers of A. suum larvae found in the lungs and livers of each group of five mice was computed. ANOVA was used to test for the effect of each magnesium diet on the dependent variables measured. LPL levels, eosinophil numbers, and total numbers of larvae per lungs or liver were compared between each group mean of different magnesium diets using the Student r test (12). Groups of mice on the various magnesium diets were compared to mice on the control diets. A value of 6 0.05 was considered significant. RESULTS BALB/c mice were placed on varying magnesium diets and infected with A. suum eggs. At 2 and 7 days pi, mice were killed and their lungs were assayed for numbers of A. suum larvae, numbers of eosinophils, and for LPL activity. One set of mice was immunized with a previous A. suum infection; a second set was not immunized. Body and Organ The total body within groups of mice at different
Weights weights or lung or liver weights were not significantly different mice receiving varying magnesium diets or between groups of pi times (Table 1).
98
HAROLD
E. LAUBACH
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TO MAGNESIUM
DEFICIENCY
99
Larvae/Lungs
At 2 days pi, the numbers of A. suum larvae found in the lungs of nonimmunized mice receiving O.O- or 200.0-g magnesium diets were found to be greater than those of the control group (Table 2). The numbers of larvae/lungs of nonimmunized mice were greater at 7 days pi when compared to those at 2 days pi. Immunized mice on O.O- or 200.0-g magnesium diets had increased numbers of larvae/lungs at 2 days pi when compared to the control group: at 7 days pi, the opposite effect was seen. Numbers of larvae per lungs of immunized mice at 2 days pi significantly decreased at 7 days pi. Numbers of larvae/lungs were lower in nonimmunized mice when compared to immunized mice at 2 days pi but were higher in nonimmunized mice 7 days pi when compared to immunized mice. Larvae/Liver
The numbers of A. ~uum larvae in the liver tissues of nonimmunized mice were not different from those in the control group at 2 days pi (Table 3). Numbers of larvae in nonimmunized mice on O.O- or 200.0-g magnesium diets were significantly higher at 7 days pi than the numbers of larvae in the livers of mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets. Numbers of larvae/livers were higher in nonimmunized mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets at 2 days pi when compared to 7 days pi. Numbers of larvae found in the livers of nonimmunized mice at 2 days pi were not different from those of immunized mice. At 7 days pi, nonimmunized mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had greater numbers of larvae than immunized mice receiving the same diets. EosinophilslLungs
Eosinophil numbers were consistent in the lungs of all nonimmunized mice when compared to the control group, at 2 days pi (Table 2). Immunized mice had lowered eosinophil numbers in groups receiving O.O- or 200.0-g magnesium diets when compared to the control group, at 2 or 7 days pi. Immunized mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had increased eosinophils/lungs when compared to the nonimmunized mice on the same diets. EosinophilsjLiver
Eosinophils/liver were not different from control groups of mice at 2 days pi with A. suum eggs and also at 7 days pi (Table 3). Eosinophil numbers increased in the liver tissues of nonimmunized mice 7 days pi when compared to 2 days pi. At 2 and 7 days pi, immunized mice receiving O.O- or 200.0-g magnesium diets had increased eosinophils in liver tissues when compared to the 400.0-g magnesium diet group (control), respectively for each set. Eosinophil numbers decreased at 7 days pi in immunized mice on 400.0 (control)-, 600.0-, or 800.0g magnesium diets when compared to the same diet groups at 2 days pi. At 2 days pi, all groups of nonimmunized mice had lower numbers of eosinophils in liver tissues than immunized mice. At 7 days pi, immunized mice receiving O.Oor 200.0-g magnesium diets had increased numbers of eosinophils/liver tissues
16’-‘.‘.’ 13”.b.C 4b.’ 3b.’ 4b.L 3.2 3.3 18.4 17.9 18.3
3.1 3.4 2.80 3.0 3.2 t + ? 2 -c
+ 5 ? 2 + 0.5” 0.4” 1.9’ 2.3” 1.8’
0.6 0.4 0.5 0.7 0.3 1210 1420 2450 2180 2150
780 840 220 240 190
53”,’ 51”.’ 22” 27 20”
+ 61”.‘.’ + W.b.’ r+ 109’.’ 2 84’.’ + 97b.’
2 f t a 2
LPL activity 2 f -c 2 2
18”.b.’ 16”.‘.’ 11“’ 12“’ lob,1
7 f lb.’ 8 2 I’.’ 6 & lb,’ 5 + lb.’ ,j & zh.’
295 193 118 124 122
Larval numbers
3.6 3.1 15.7 12.5 14.2
2.9 3.4 3.2 3.0 3.1
2 -c + 2 -e
k k 2 t +
0.5” 0.3” 1.6 1.2” 1.4
0.4 0.4 0.6 0.5 0.7
Eosinophils
7 days postinfection
240 130 210 180 210
840 830 820 810 830
f f + + r
k + f -c 2
31b.’ 27’.’ 16’,’ 12b.’ 196J
66’ 67’ 74’,’ 64b.’ 70*.’
LPL activity
means from hve mice ? 1 standard deviation. Numbers of eosinophils for each tissue sample were expressed counted. Comparing the differences between sample means, a denotes P s 0.05 when the control group was the same set; b denotes P s 0.05 between 2 and 7 days pi; and c denotes P G 0.05 between nonimmunized magnesium diets.
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Eosinophils
2 days postinfection
TABLE 2 on Numbers of Larvae, Eosinophils, and LPL Activity in Mouse Lungs after Infection with Ascaris mum
Larval numbers
Note. The values represent the as an average of ten 400x fields compared to another group within and immunized mice on the same
A. Nonimmunized mice 0.0 200.0 400.0 (control) 600.0 800.0 B. Immunized mice 0.0 200.0 400.0 (control) 600.0 800.0
Dietary groups (g W#WW per 100 kg diet)
Effect of Dietary Magnesium
2
X % g ‘3 F” g
z 0
mice 0.4” 2k 0.1* -t 0.3h -r- 0.4h ? 0.7h
6.4 2 0.9h.’
16.1 2.1”,’ 15.3 6.2 f”2 0.8”.’ 2.2-l 6.6 k l.Oh.’
2.9 3.4 3.2 3.2 3.1
Eosinophils
2 days postinfection
1050 k 48”.’
1430 It2 51”.’ 69”,“.’ 1010 1460 62”.b.” 980 zt 43h.’
960278 940 f 70 220 + 22h 190 f 19b 180 ? 166
LPL activity
Note. The results are expressed in the same manner as those in Table 2.
112 c 16’
11 10 7b 9’ lob
800.0
2f f !z It
96 2k+- 17 14 112 93 12” 109 f 14b
119 116 102 98 103
Larval numbers
0.0 400.0 200.0 (control) 600.0
0.0 200.0 400.0 (control) 600.0 800.0 B. Immunized mice
A. Nonimmunized
Dietary groups 6s WQ/H,O per 100 kg diet)
78 18” lob.’ 136.’ 96.’
7 2 lb.’
719 f+2 9” 12” 64 2D,’ 12 _t 2h.’
96 ff 110 45 2 47 2 51 t
Larval numbers
ff _’ f ?
l.lb.’ 0.86.’ 1.2’.‘ 0.8’.’ l.O”,’
3.7 k 0.6’,’
17.8 2.4”,’ 18.5 3.6 i+” 0.3h.’ 1.9”.’ 3.3 _’ 0.56.‘
11.2 10.7 9.8 10.1 10.6
Eosinophils
7 days postinfection
suum
420 + 35’.’
850 71”,b,’ 420 910 2-c f 38’.’ 7W,b,C 390 -t 36’.’
1120?81 990%72 920 2 74b,’ 1110 f 796,’ 1230 2 86’.’
LPL activity
TABLE 3 Effect of Dietary Magnesium on Numbers of Larvae, Eosinophils, and LPL Activity in Mouse Livers after Infection with Ascnris
z Fj i;j
5 E g 2
z
% 2 g
rz
102
HAROLD
E. LAUBACH
when compared to the same groups of nonimmunized mice. Immunized mice on 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had lower numbers of eosinophils than the same diet groups of nonimmunized mice. LPL Activity/Lungs LPL activity in the lungs of nonimmunized mice, 2 days pi, was increased in mice receiving O.O- or 200.0-g magnesium diets when compared to the control group; there was not a difference within groups at 7 days pi (Table 2). Nonimmunized mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had lower LPL activity at 2 days pi than at 7 days pi. Immunized mice, at 2 days pi, had low levels of lung LPL activity in groups receiving O.O- or 200.0g magnesium when compared to the control group; all groups of immunized mice had consistent LPL activity levels at 7 days pi. LPL activity levels of immunized mouse groups were decreased at 7 days pi when compared to groups at 2 days pi. All immunized mouse groups had increased lung LPL activity at 2 days pi when compared to nonimmunized mice but had lower LPL activity at 7 days pi. LPL Activity/Liver LPL activity of liver tissue from nonimmunized mice on O.O- or 200-g magnesium diets was increased at 2 days pi with A. suum eggs when compared to the control group; this difference was not seen at 7 days pi (Table 3). Nonimmunized mice on 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had lower LPL activities in their livers at 2 days pi than at 7 days pi. Immunized mice on O.O- or 200.0-g magnesium diets had increased LPL activities at 2 days and 7 days pi, respectively, when compared to the control groups. Increased LPL activity in liver tissue was evident in all groups of immunized mice at 2 days pi when compared to the same groups at 7 days pi. At 2 days pi, all groups of immunized mice had increased LPL activity in liver tissue when compared to groups of nonimmunized mice. At 7 days pi, immunized mice on 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had lower LPL activity in liver tissue than the same groups of nonimmunized mice. DISCUSSION
A. suum infections in mice are characterized by a temporal change in the distribution of larvae in the lungs and liver (13). The numbers of larvae found in these organs are influenced by the challenge dose and by the immune status of the mice. This host response to infection was used to demonstrate the effect of alterations in magnesium diets on migration of A. suum larvae through the lungs and livers of mice and the subsequent development of a tissue eosinophilia or an increased tissue LPL activity. Previous studies demonstrate that there is a relationship between tissue eosinophil numbers and LPL activity (4-7) and Tcell regulation of this process (8). Magnesium deficiency has a direct effect on T-cell responses in mice (3). In this study, nonimmunized mice had increased numbers of larvae/lungs at 2 days and at 7 days pi in O.O- and 200.0-g magnesium diet groups with increased
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103
LPL only in the 2-day-pi group suggesting a direct effect of dietary magnesium on the removal of larvae from the lungs (Table 2). There was an increased LPL activity at 2 days pi but not at 7 days pi, when numbers of larvae were significantly increased from those at 2 days pi, proposing a suppression of LPL activity with increased larvae/lungs. Eosinophil numbers were not different between these groups of nonimmunized mice demonstrating the absence of a tissue eosinophilia with increased numbers of larvae and an inadequate correlation of eosinophils to LPL activity at 2 days pi. Numbers of larvae were increased in 0- and 200-g magnesium diet groups of immunized mice at 2 days pi accompanied by decreased eosinophil numbers and LPL activity (Table 2). Numbers of larvae/lungs were significantly decreased at 7 days pi with decreased eosinophil numbers in mice on O.O- and 200.0-g magnesium diets; LPL was low for all groups. These data propose that increased tissue eosinophil numbers are dependent on the presence of sufficient magnesium in the diet of immunized mice and that LPL activity is not affected by increased numbers of tissue eosinophils. Liver tissue responses of nonimmunized mice were characterized by consistent numbers of A. suum larvae and tissue eosinophils at 2 days pi demonstrating the absence of significant changes with regard to dietary magnesium differences (Table 3). LPL activity was significantly increased in mice recieving O.O- or 200.0-g magnesium diets, a finding that did not correlate with numbers of larvae or eosinophils/liver suggesting an independent effect of dietary magnesium on LPL activity. At 7 days pi, larvae/liver were increased in nonimmunized mouse groups receiving O.O- or 200.0-g magnesium diets without changes in numbers of eosinophils in the liver tissue or LPL activity. These data demonstrate a loss of the ability of liver tissue to respond to A. suum larvae when mice are on low magnesium diets. Immunized mice had consistent numbers of larvae/liver tissue at 2 days pi with increased numbers of tissue eosinophils and increased tissue LPL activity in groups receiving O.O- or 200.0-g magnesium diets (Table 3). These data suggest an independent effect of dietary magnesium on eosinophil numbers and LPL activity not associated with numbers of larvae/liver. A. suum larvae/liver, eosinophils/liver, and LPL activity were increased at 7 days pi in mice receiving O.O- or 200.0-g magnesium diets implying a correlation between these findings and the immune response to infection. SUMMARY
Low amounts of dietary magnesium affected the inflammatory tissue response in nonimmunized mice differently than in immunized mice. Eosinophil numbers and LPL activity in lung tissue following infection with A. wum larvae were altered by the level of magnesium in the diets of mice. Average or higher dietary levels of magnesium resulted in decreased numbers of lung larvae indicating an overall protective effect. Increases in eosinophil numbers or LPL activity were not directly related to the numbers of larvae/lungs. Larvae/livers, eosinophil numbers, and LPL activity were affected by the types of magnesium diets that mice received. Nonimmunized mice had differences in larvae/liver (at 2 days
104
HAROLD
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and 7 days pi) and LPL activity (at 2 days pi). Immunized mice had varying findings at 2 days pi but a direct relationship between dietary magnesium and numbers of larvae, numbers of eosinophils, and liver LPL activity at 7 days pi. ACKNOWLEDGMENTS This investigation was supported by Grant 81-15-008 from the American Osteopathic Association. I thank Lee Volker for her excellent clerical help.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Kashiwa, H. K., and Hungerford, G. F., Proc. Sot. hp. Biol. Med. 99, 441 (1958). Whang, R., and Welt, L. G., J. Clin. Invest. 42, 305 (1963). Guenounou, M., Armier, J., and Gaudin-Harding, F., Znt. J. Vit. Nutr. Res. 48, 290 (1978). Laubach, H., Kocan, A. A., and Sartain, K. E., .Z. Purasitol. 64, 1145 (1978). Ottolenghi, A., Weatherly, N. F., Kocan, A. A., and Larsh. J. E., Jr., Infect. Zmmun. 15, 13 (1977). Ottolenghi, A., Kocan, A. A., Weatherly, N. F., and Larsh, J. E., Jr., Exp. Par&to/. 38, 96 (1975). Goulson, H. T., Ottolenghi, A., and Larsh, J. E., Jr., Amer. J. Trap. Med. Hyg. 30, 350 (1981). Goven, A. J., and Moore, G. W., 2. Purasitenkd. 61, 265 (1980). Caster, W. O., and Bleecher, S., .Z. Nutr. 105, 308 (1975). Fairbaim, D., Canad. .Z. Zool. 39, 153 (1961). Ottolenghi, A., Lipids 8, 426 (1973). Snedecor, G. W., and Cochran, W. G., in “Statistical Methods,” p. 59. Iowa State Univ. Press, Ames, IA, 1972. Bindseil, E., Acta Puthol. Microbial. &and. 77, 223 (1969).
MEDICINE
AND
METABOLIC
BIOLOGY
42, 95-104 (1989)
Effect of Dietary Magnesium on Ascaris suum infections of Mice HAROLD
E.
LAUBACH
Department of Microbiology, Southeastern University of the Health Sciences, 1750 N. E. 168th Street, North Miami Beach, Florida 33162 Received January 26, 1989, and in revised form June 1, 1989
Magnesium deficiency in mammals is characterized in its earliest stage by a series of clinical and laboratory manifestations, such as a pronounced hyperemia of the skin, leukocytosis, and eosinophilia (1,2). These characteristics develop in 6 to 9 days and are suggestive of a transient hypersensitive state. If animals are sacrificed after 21 to 28 days on a magnesium deficient diet, many microscopic lesions are found in various tissues. The lesions are characterized by focal perivascular collections of inflammatory cells which may be associated with cell necrosis. The histological picture resembles that seen in delayed hypersensitivity reactions. A deficiency in dietary magnesium has been shown to have a marked effect on the immune response in mice (3). Mice on a magnesium-deficient diet were immunized with sheep red blood cells and the immune response was studied by estimating the number of spleen antibody-forming cells capable of lysing the red blood cells and by a cytoadhesion test to determine the number of rosette-forming cells, T lymphocytes. Magnesium deficiency produced a drastic fall in the primary and secondary immune responses, as measured by the number of spleen antibodyforming cells. The number of rosette-forming cells was also much lower in the spleens of magnesium-deficient mice. With these present data on magnesium deficiency, the question was posed as to its effect on the inflammatory response to helminth infections. Previous research has demonstrated that there are increased levels of lysophospholipase (LPL: EC 3.1.1 S) in the lungs of animals infected with Angiostrongyhs cantonensis (4,5), Nippostrongylus brusiliensis(6), and Strongyloides ratti (7). Increased LPL activity correlates with increased tissue eosinophilia and with the presence of the parasites in the tissues. An anamestic response was observed. As it has been shown that T-cell numbers are reduced when mice are placed on magnesium-deficient diets (3) and that T lymphocytes are involved in LPL activity (8), it would be interesting to study the synergistic action of magnesium deficiency on changes in the inflammatory response to infection with Ascaris suum in mice. A model was used that included A. suum infection as the inducer of inflammation 95 0885-4505189 $3.00 Copyright Q 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.
96
HAROLD
E. LAUBACH
and the effect of magnesium deficiency was assessed by measuring the numbers of eosinophils and larvae found in liver or lung tissues and the LPL activity associated with these infected organs. MATERIALS
AND METHODS
Male, weanling BALB/c AnNCr/BR Mice (Charles River Breeding Laboratories, Wilmington, MA) were divided into 20 groups of 10 mice each and fed defined diets. The mice were maintained at 25°C in a well-ventilated animal room. Mice were divided into four sets, each with five groups of 10 mice each. One group of 10 mice in each of the four sets of mice was dietary magnesium control mice. Control groups of mice received a nutritionally defined diet consisting of 16% casein, 4% lactalbumin, 4% cellulose, 4% of a complete vitamin mixture (9), 48% wheat starch, 16% sucrose, 4% corn oil, and a complete mineral oil mixture (699 g CaCO,, 966 g KH2P0,, 1220 g CaHOP,, 400 g MgS0,J7H20, 508 g NaCl, 191 g Na,Se03, 0.03 g KIO,, 17.3 g MnS04/H20, 2.2 g CuS04/4H20, 2.6 g ZnCo,, 1.2 g KZCr207, 0.003 g NaF, and 10.4 g FeS0,/7Hz0 per loo-kg diet). This defined diet was supplied in sufficient quantity so that the mice could eat unlimited quantities. Mice were also given demineralized water in unlimited quantities, The experimental magnesium diet groups of mice received the defined diet modified in a way to provide 0.0, 200.0, 600.0, or 800.0 g MgS04/7HZ0 per 100 kg diet. These mice were also given their diets and demineralized water in unlimited quantities. Viable eggs of female A. suum worms were digested out of the distal third of the uterus using NaOH, washed with buffered saline, and placed into petri dishes in dilute sulfuric acid. The eggs were allowed to embryonate and were then washed with buffered saline prior to use (10). Mice were inoculated, per OS, with 1000 A. suum infective eggs by means of a dosing needle inserted into the esophagus. Groups of mice were infected 21 days after they were placed on their various magnesium diets. Control mouse groups were infected in the same manner as the experimental mouse groups. The four sets of animals consisted of two sets of mice immunized with A. suum infective eggs and two sets of nonimmunized mice. The first set of mice were nonimmunized mice that were challenged with 1000 A. suum eggs. The second set were also nonimmunized mice but were not challenged with eggs. The third set were immunized mice and were challenged with 1000 A. suum eggs. The fourth set of mice were immunized but were not challenged with eggs. Immunized mice were infected with 1000 A. suum infective eggs, per OS, 30 days before challenge with 1000 A. suum eggs. Ali four sets of mice had five mice per group killed 2 days after challenge with eggs and the remaining five mice in each group were killed 7 days postinfection (pi). At necropsy, total body and blotted lung and liver weights were recorded. One portion of lung and liver tissue from each mouse was crushed with a tissue grinder and digested in a solution of 0.7% pepsin and 1.0% hydrochloric acid. The digest was searched for larvae, in small volumes in a petri dish, until all of
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TO MAGNESIUM
DEFICIENCY
97
the digest was examined. Total larvae present in the lungs and liver of each mouse were recorded. A second portion of liver and lung tissue from each mouse was fixed in 10% buffered formalin immediately after death. Tissue samples were stained with hematoxylin-eosin, embedded in paraffin, sectioned, and mounted onto slides. Stained sections were examined for pathological changes at 400X using brightfield illumination. Cell types in the area of A. suum larvae, but not adjacent to the larvae, were observed for each tissue preparation. A total of 10 fields was examined for each tissue sample and expressed as average counts per field at 400X illumination. A third portion of lung and liver tissue from each mouse was assayed for LPL activity according to the method of Ottolenghi (11). Lung and liver tissues were weighed, mixed I:20 with cold 12.5% glycerol solution, and ground up. Fivemicroliter samples of the mixtures were added to each of four test tubes and equilibrated in a 37°C bath for 4 min. Three-tenths milliliter of prewarmed lysophosphatidylcholine (substrate for LPL) solution (20 kl/ml) was added to each of the test tubes and incubated at 37°C for 30 min. Control tubes consisted of four 5~1 samples of the lung and liver mixtures without lysophosphatidylcholine solution added. The enzyme reactions were stopped by the addition of 0.1 ml of 2 NH2S04, 1.0 ml of isopropyl alcohol, and 0.4 ml of water. Two milliliters of heptane was added to each tube and mixed. The heptane was removed and titrated with 0.01 N NaOH and bromthymol blue while bubbling nitrogen into the test tube. LPL activity was expressed as micromoles per gram tissue/per hour. The average & standard deviation of the body weight and lung and liver weights, LPL levels, eosinophil numbers, and the numbers of A. suum larvae found in the lungs and livers of each group of five mice was computed. ANOVA was used to test for the effect of each magnesium diet on the dependent variables measured. LPL levels, eosinophil numbers, and total numbers of larvae per lungs or liver were compared between each group mean of different magnesium diets using the Student r test (12). Groups of mice on the various magnesium diets were compared to mice on the control diets. A value of 6 0.05 was considered significant. RESULTS BALB/c mice were placed on varying magnesium diets and infected with A. suum eggs. At 2 and 7 days pi, mice were killed and their lungs were assayed for numbers of A. suum larvae, numbers of eosinophils, and for LPL activity. One set of mice was immunized with a previous A. suum infection; a second set was not immunized. Body and Organ The total body within groups of mice at different
Weights weights or lung or liver weights were not significantly different mice receiving varying magnesium diets or between groups of pi times (Table 1).
98
HAROLD
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RESPONSE
TO MAGNESIUM
DEFICIENCY
99
Larvae/Lungs
At 2 days pi, the numbers of A. suum larvae found in the lungs of nonimmunized mice receiving O.O- or 200.0-g magnesium diets were found to be greater than those of the control group (Table 2). The numbers of larvae/lungs of nonimmunized mice were greater at 7 days pi when compared to those at 2 days pi. Immunized mice on O.O- or 200.0-g magnesium diets had increased numbers of larvae/lungs at 2 days pi when compared to the control group: at 7 days pi, the opposite effect was seen. Numbers of larvae per lungs of immunized mice at 2 days pi significantly decreased at 7 days pi. Numbers of larvae/lungs were lower in nonimmunized mice when compared to immunized mice at 2 days pi but were higher in nonimmunized mice 7 days pi when compared to immunized mice. Larvae/Liver
The numbers of A. ~uum larvae in the liver tissues of nonimmunized mice were not different from those in the control group at 2 days pi (Table 3). Numbers of larvae in nonimmunized mice on O.O- or 200.0-g magnesium diets were significantly higher at 7 days pi than the numbers of larvae in the livers of mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets. Numbers of larvae/livers were higher in nonimmunized mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets at 2 days pi when compared to 7 days pi. Numbers of larvae found in the livers of nonimmunized mice at 2 days pi were not different from those of immunized mice. At 7 days pi, nonimmunized mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had greater numbers of larvae than immunized mice receiving the same diets. EosinophilslLungs
Eosinophil numbers were consistent in the lungs of all nonimmunized mice when compared to the control group, at 2 days pi (Table 2). Immunized mice had lowered eosinophil numbers in groups receiving O.O- or 200.0-g magnesium diets when compared to the control group, at 2 or 7 days pi. Immunized mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had increased eosinophils/lungs when compared to the nonimmunized mice on the same diets. EosinophilsjLiver
Eosinophils/liver were not different from control groups of mice at 2 days pi with A. suum eggs and also at 7 days pi (Table 3). Eosinophil numbers increased in the liver tissues of nonimmunized mice 7 days pi when compared to 2 days pi. At 2 and 7 days pi, immunized mice receiving O.O- or 200.0-g magnesium diets had increased eosinophils in liver tissues when compared to the 400.0-g magnesium diet group (control), respectively for each set. Eosinophil numbers decreased at 7 days pi in immunized mice on 400.0 (control)-, 600.0-, or 800.0g magnesium diets when compared to the same diet groups at 2 days pi. At 2 days pi, all groups of nonimmunized mice had lower numbers of eosinophils in liver tissues than immunized mice. At 7 days pi, immunized mice receiving O.Oor 200.0-g magnesium diets had increased numbers of eosinophils/liver tissues
16’-‘.‘.’ 13”.b.C 4b.’ 3b.’ 4b.L 3.2 3.3 18.4 17.9 18.3
3.1 3.4 2.80 3.0 3.2 t + ? 2 -c
+ 5 ? 2 + 0.5” 0.4” 1.9’ 2.3” 1.8’
0.6 0.4 0.5 0.7 0.3 1210 1420 2450 2180 2150
780 840 220 240 190
53”,’ 51”.’ 22” 27 20”
+ 61”.‘.’ + W.b.’ r+ 109’.’ 2 84’.’ + 97b.’
2 f t a 2
LPL activity 2 f -c 2 2
18”.b.’ 16”.‘.’ 11“’ 12“’ lob,1
7 f lb.’ 8 2 I’.’ 6 & lb,’ 5 + lb.’ ,j & zh.’
295 193 118 124 122
Larval numbers
3.6 3.1 15.7 12.5 14.2
2.9 3.4 3.2 3.0 3.1
2 -c + 2 -e
k k 2 t +
0.5” 0.3” 1.6 1.2” 1.4
0.4 0.4 0.6 0.5 0.7
Eosinophils
7 days postinfection
240 130 210 180 210
840 830 820 810 830
f f + + r
k + f -c 2
31b.’ 27’.’ 16’,’ 12b.’ 196J
66’ 67’ 74’,’ 64b.’ 70*.’
LPL activity
means from hve mice ? 1 standard deviation. Numbers of eosinophils for each tissue sample were expressed counted. Comparing the differences between sample means, a denotes P s 0.05 when the control group was the same set; b denotes P s 0.05 between 2 and 7 days pi; and c denotes P G 0.05 between nonimmunized magnesium diets.
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Eosinophils
2 days postinfection
TABLE 2 on Numbers of Larvae, Eosinophils, and LPL Activity in Mouse Lungs after Infection with Ascaris mum
Larval numbers
Note. The values represent the as an average of ten 400x fields compared to another group within and immunized mice on the same
A. Nonimmunized mice 0.0 200.0 400.0 (control) 600.0 800.0 B. Immunized mice 0.0 200.0 400.0 (control) 600.0 800.0
Dietary groups (g W#WW per 100 kg diet)
Effect of Dietary Magnesium
2
X % g ‘3 F” g
z 0
mice 0.4” 2k 0.1* -t 0.3h -r- 0.4h ? 0.7h
6.4 2 0.9h.’
16.1 2.1”,’ 15.3 6.2 f”2 0.8”.’ 2.2-l 6.6 k l.Oh.’
2.9 3.4 3.2 3.2 3.1
Eosinophils
2 days postinfection
1050 k 48”.’
1430 It2 51”.’ 69”,“.’ 1010 1460 62”.b.” 980 zt 43h.’
960278 940 f 70 220 + 22h 190 f 19b 180 ? 166
LPL activity
Note. The results are expressed in the same manner as those in Table 2.
112 c 16’
11 10 7b 9’ lob
800.0
2f f !z It
96 2k+- 17 14 112 93 12” 109 f 14b
119 116 102 98 103
Larval numbers
0.0 400.0 200.0 (control) 600.0
0.0 200.0 400.0 (control) 600.0 800.0 B. Immunized mice
A. Nonimmunized
Dietary groups 6s WQ/H,O per 100 kg diet)
78 18” lob.’ 136.’ 96.’
7 2 lb.’
719 f+2 9” 12” 64 2D,’ 12 _t 2h.’
96 ff 110 45 2 47 2 51 t
Larval numbers
ff _’ f ?
l.lb.’ 0.86.’ 1.2’.‘ 0.8’.’ l.O”,’
3.7 k 0.6’,’
17.8 2.4”,’ 18.5 3.6 i+” 0.3h.’ 1.9”.’ 3.3 _’ 0.56.‘
11.2 10.7 9.8 10.1 10.6
Eosinophils
7 days postinfection
suum
420 + 35’.’
850 71”,b,’ 420 910 2-c f 38’.’ 7W,b,C 390 -t 36’.’
1120?81 990%72 920 2 74b,’ 1110 f 796,’ 1230 2 86’.’
LPL activity
TABLE 3 Effect of Dietary Magnesium on Numbers of Larvae, Eosinophils, and LPL Activity in Mouse Livers after Infection with Ascnris
z Fj i;j
5 E g 2
z
% 2 g
rz
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HAROLD
E. LAUBACH
when compared to the same groups of nonimmunized mice. Immunized mice on 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had lower numbers of eosinophils than the same diet groups of nonimmunized mice. LPL Activity/Lungs LPL activity in the lungs of nonimmunized mice, 2 days pi, was increased in mice receiving O.O- or 200.0-g magnesium diets when compared to the control group; there was not a difference within groups at 7 days pi (Table 2). Nonimmunized mice receiving 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had lower LPL activity at 2 days pi than at 7 days pi. Immunized mice, at 2 days pi, had low levels of lung LPL activity in groups receiving O.O- or 200.0g magnesium when compared to the control group; all groups of immunized mice had consistent LPL activity levels at 7 days pi. LPL activity levels of immunized mouse groups were decreased at 7 days pi when compared to groups at 2 days pi. All immunized mouse groups had increased lung LPL activity at 2 days pi when compared to nonimmunized mice but had lower LPL activity at 7 days pi. LPL Activity/Liver LPL activity of liver tissue from nonimmunized mice on O.O- or 200-g magnesium diets was increased at 2 days pi with A. suum eggs when compared to the control group; this difference was not seen at 7 days pi (Table 3). Nonimmunized mice on 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had lower LPL activities in their livers at 2 days pi than at 7 days pi. Immunized mice on O.O- or 200.0-g magnesium diets had increased LPL activities at 2 days and 7 days pi, respectively, when compared to the control groups. Increased LPL activity in liver tissue was evident in all groups of immunized mice at 2 days pi when compared to the same groups at 7 days pi. At 2 days pi, all groups of immunized mice had increased LPL activity in liver tissue when compared to groups of nonimmunized mice. At 7 days pi, immunized mice on 400.0 (control)-, 600.0-, or 800.0-g magnesium diets had lower LPL activity in liver tissue than the same groups of nonimmunized mice. DISCUSSION
A. suum infections in mice are characterized by a temporal change in the distribution of larvae in the lungs and liver (13). The numbers of larvae found in these organs are influenced by the challenge dose and by the immune status of the mice. This host response to infection was used to demonstrate the effect of alterations in magnesium diets on migration of A. suum larvae through the lungs and livers of mice and the subsequent development of a tissue eosinophilia or an increased tissue LPL activity. Previous studies demonstrate that there is a relationship between tissue eosinophil numbers and LPL activity (4-7) and Tcell regulation of this process (8). Magnesium deficiency has a direct effect on T-cell responses in mice (3). In this study, nonimmunized mice had increased numbers of larvae/lungs at 2 days and at 7 days pi in O.O- and 200.0-g magnesium diet groups with increased
RESPONSE
TO MAGNESIUM
DEFICIENCY
103
LPL only in the 2-day-pi group suggesting a direct effect of dietary magnesium on the removal of larvae from the lungs (Table 2). There was an increased LPL activity at 2 days pi but not at 7 days pi, when numbers of larvae were significantly increased from those at 2 days pi, proposing a suppression of LPL activity with increased larvae/lungs. Eosinophil numbers were not different between these groups of nonimmunized mice demonstrating the absence of a tissue eosinophilia with increased numbers of larvae and an inadequate correlation of eosinophils to LPL activity at 2 days pi. Numbers of larvae were increased in 0- and 200-g magnesium diet groups of immunized mice at 2 days pi accompanied by decreased eosinophil numbers and LPL activity (Table 2). Numbers of larvae/lungs were significantly decreased at 7 days pi with decreased eosinophil numbers in mice on O.O- and 200.0-g magnesium diets; LPL was low for all groups. These data propose that increased tissue eosinophil numbers are dependent on the presence of sufficient magnesium in the diet of immunized mice and that LPL activity is not affected by increased numbers of tissue eosinophils. Liver tissue responses of nonimmunized mice were characterized by consistent numbers of A. suum larvae and tissue eosinophils at 2 days pi demonstrating the absence of significant changes with regard to dietary magnesium differences (Table 3). LPL activity was significantly increased in mice recieving O.O- or 200.0-g magnesium diets, a finding that did not correlate with numbers of larvae or eosinophils/liver suggesting an independent effect of dietary magnesium on LPL activity. At 7 days pi, larvae/liver were increased in nonimmunized mouse groups receiving O.O- or 200.0-g magnesium diets without changes in numbers of eosinophils in the liver tissue or LPL activity. These data demonstrate a loss of the ability of liver tissue to respond to A. suum larvae when mice are on low magnesium diets. Immunized mice had consistent numbers of larvae/liver tissue at 2 days pi with increased numbers of tissue eosinophils and increased tissue LPL activity in groups receiving O.O- or 200.0-g magnesium diets (Table 3). These data suggest an independent effect of dietary magnesium on eosinophil numbers and LPL activity not associated with numbers of larvae/liver. A. suum larvae/liver, eosinophils/liver, and LPL activity were increased at 7 days pi in mice receiving O.O- or 200.0-g magnesium diets implying a correlation between these findings and the immune response to infection. SUMMARY
Low amounts of dietary magnesium affected the inflammatory tissue response in nonimmunized mice differently than in immunized mice. Eosinophil numbers and LPL activity in lung tissue following infection with A. wum larvae were altered by the level of magnesium in the diets of mice. Average or higher dietary levels of magnesium resulted in decreased numbers of lung larvae indicating an overall protective effect. Increases in eosinophil numbers or LPL activity were not directly related to the numbers of larvae/lungs. Larvae/livers, eosinophil numbers, and LPL activity were affected by the types of magnesium diets that mice received. Nonimmunized mice had differences in larvae/liver (at 2 days
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and 7 days pi) and LPL activity (at 2 days pi). Immunized mice had varying findings at 2 days pi but a direct relationship between dietary magnesium and numbers of larvae, numbers of eosinophils, and liver LPL activity at 7 days pi. ACKNOWLEDGMENTS This investigation was supported by Grant 81-15-008 from the American Osteopathic Association. I thank Lee Volker for her excellent clerical help.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Kashiwa, H. K., and Hungerford, G. F., Proc. Sot. hp. Biol. Med. 99, 441 (1958). Whang, R., and Welt, L. G., J. Clin. Invest. 42, 305 (1963). Guenounou, M., Armier, J., and Gaudin-Harding, F., Znt. J. Vit. Nutr. Res. 48, 290 (1978). Laubach, H., Kocan, A. A., and Sartain, K. E., .Z. Purasitol. 64, 1145 (1978). Ottolenghi, A., Weatherly, N. F., Kocan, A. A., and Larsh. J. E., Jr., Infect. Zmmun. 15, 13 (1977). Ottolenghi, A., Kocan, A. A., Weatherly, N. F., and Larsh, J. E., Jr., Exp. Par&to/. 38, 96 (1975). Goulson, H. T., Ottolenghi, A., and Larsh, J. E., Jr., Amer. J. Trap. Med. Hyg. 30, 350 (1981). Goven, A. J., and Moore, G. W., 2. Purasitenkd. 61, 265 (1980). Caster, W. O., and Bleecher, S., .Z. Nutr. 105, 308 (1975). Fairbaim, D., Canad. .Z. Zool. 39, 153 (1961). Ottolenghi, A., Lipids 8, 426 (1973). Snedecor, G. W., and Cochran, W. G., in “Statistical Methods,” p. 59. Iowa State Univ. Press, Ames, IA, 1972. Bindseil, E., Acta Puthol. Microbial. &and. 77, 223 (1969).