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Effect of zinc deficiency on autologous rosette-forming cells
CELLULAR
IMMUNOLOGY
48, 238-243
(1979)
Effect of Zinc Deficiency LEONARD
Memorial
NASH,
on Autologous
Rosette-Forming
TSUTOMLJ IWATA, GABRIEL FERNANDES, AND GENEVIEVE S. INCEFY’
Sloan-Kettering
Cancer
Center, Received
1275 York Avenue, April
New
ROBERT
York,
New
Cells A. GOOD,
York
10021
25, 1979
Ability to form autologous rosettes (A-rosettes) is characteristic of a certain population of immature T cells, present in the thymus of various species including man. In mice, few A-rosettes are normally found in peripheral blood and spleen but their number increases markedly in spleen following thymectomy. In our studies, A-rosette formation could be demonstrated to be significantly enhanced in the spleen of C57Bl/Ks mice after the animals had been maintained 3,6, and 8 weeks on a zinc-deficient diet (Zn-) and to increase progressively with duration of feeding the Zn diet. These changes were quickly reversed by feeding a normal diet containing zinc but could not be eliminated by complete adrenalectomy, a finding that ruled out their dependence on pituitary-adrenal as is function attributable to stress. Pair-fed controls and mice fed a zinc-supplemented diet ad l&turn showed few A-rosettes, as expected.
INTRODUCTION The maturation of lymphoid stem cells into peripheral T lymphocytes involves several stages of development. An initial step appears to depend on direct interaction with epithelium in vitro during passage of precursors through the thymus while subsequent stages of differentiation are attributable to influence of thymic hormones or circulating serum thymic factors (l-4). Ability to form autologous rosettes (A-rosettes) is characteristic of a certain population of immature T cells, which have been demonstrated in the thymus of various species, including man (5, 6). In mice, few A-rosettes are normally present in peripheral blood and spleen, but such cells are markedly increased in the spleen following thymectomy (7). Treatment with thymic hormone in vitro reduces the number of A-rosettes in the blood and spleen of thymectomized mice (7). Zinc, an essential metal for maximum activity of many enzymes (8), has also been demonstrated to be necessary for the development and maintenance of the thymus and for providing normal levels of circulating serum thymic factor, “facteur thymique serique” (FTS) (9). It was of interest, therefore, to study what influence Zn deficiency might have on such a population of precursor or immature T cells, and to attempt to correlate changes in number of A-rosette-forming cells (A-RFC) with the severity of Zn deficiency. A-rosette formation could be demonstrated to be enhanced in the spleen of C57Bl/Ks mice after the animals had been maintained 3 weeks on a Zn-deficient ’ To whom correspondence
and reprint requests should be addressed.
000%8749/79/130238-06$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
239
diet (Zn-), and more pronounced increases of A-rosettes were observed after 6 and 8 weeks on this diet. Changes in this immature T-cell population corresponded with the fall of FTS to low levels during Zn restriction. These changes were quickly reversed by feeding a normal diet containing zinc but could not be eliminated by complete adrenalectomy, a finding that ruled out their dependence on pituitaryadrenal as is function attributable to stress. Pair-fed controls and mice fed a Zn-supplemented diet (Zn’) ad libitum showed few A-rosettes, as expected. MATERIAL
AND METHODS
Animals and diets. Male and female C57Bl/Ks mice were divided into seven groups when they were 6 weeks old. Thirty-five mice were fed Purina lab chow ad libitum as controls, 50 were fed a zinc-deficient (Zn-) diet, and 50 a zinc-supplemented (Zn+) diet (Taklad test diets). Further, the Zn- and Zn+ groups were divided into adrenalectomized (Adx) and sham Adx groups of 10 mice each. The zinc content of the Purina lab chow was analyzed by atomic absorption spectrophotometry (10) and found to be 9.68 pg Zn/g. The 50 kg Zn/g were present in the Zn+ diet which was made up of egg white, dextrose, corn oil, and cellulose with vitamin and mineral supplements as described by Luecke et al. (11). No zinc was present in the Zn- diet and its composition was otherwise identical to the Zn+ diet. During the period of the study, mice were kept in stainless-steel cages, as previously described (9). They were fed ad fibitum and had free access to deionized distilled water, but had no contact with shavings or other material that could possibly contain zinc. Autologous rosette assay. The procedure utilized for A-rosette formation has been described in detail by Charreire and Bach (5, 6). In brief, it consisted of combining autologous or syngenic mouse erythrocytes (MRBC) and spleen cells (SC) in various MRBC/Sc ratios (16/l to 128/l). Scoring of rosettes was performed as previously described (5) except in certain cases (64/l, 128/l) when 1 and 1.25 ml of medium were added, respectively, to dilute the cell suspensions permitting easier reading. Adherent cells were depleted by prior incubation of the spleen cells for 90 min at 37°C before adding the MRBC. RESULTS Spontaneous A-rosette formation of spleen cells with mouse erythrocytes was analyzed at different MRBC/Sc ratios. Initially, spleen cells of C57BV6 Cum mice, 12 weeks old, that had been thymectomized 4 weeks earlier, were tested for spontaneous A-rosette formation. The results are shown in Fig. la. This analysis established the desired ratios of MRBC/Sc to be used for demonstration of A-RFC in mice kept on Zn- and Zn+ diets. These procedures were then applied to the analysis of spleen cells of C57Bl/Ks mice, 9 and 14 weeks old, that had been maintained for 3 and 8 weeks on Zn- diet to verify the optimal MRBC/Sc ratio at which A-rosettes should be formed with cells of these animals. The results were compared to those obtained with spleen cells from lCweek-old normal C57BVKs mice (Fig. lb). The number of cells able to form A-rosettes in the spleen of animals fed Zn-, Zn+, and lab chow diets for 3,6, and 8 weeks were investigated using optimum ratios of MRBC/Sc of 64/l. A-RFC in spleen of mice maintained on Zn- diet had increased
240
SHORT COMMUNICATIONS
Ii : : & 01
I
I
I
16
32
64 Mouse
1 128 RBC/Spleen
16 Cells
32 (MRBC/W
I
I
64
128
FIG. 1. Number of autologous rosette-forming cells according to mouse erythrocytes/spleen cells ratios. (a) Spleen cells of 12-week-old C57BV6 Cum mice tested 4 weeks after thymectomy, (0); normal controls, (0). (b) Spleen cells of C57BYKs mice, 9 weeks old, fed 3 weeks the Zn- diet, (Cl); 14 weeks old, fed 8 weeks the Zn- diet, (A); 14 weeks old, fed normal lab chow diet, (A) (mean of two or three determinations f SD).
significantly (Fig. 2). It can be seen that A-RFC increased progressively with duration of feeding the Zn- diet. Few A-RFC were present in the spleens of animals fed lab chow ad libitum or Zn+ diets, using the pair-feeding method. The effects of stress caused by the zinc deficiency was also analyzed. Spleen cells of mice Adx or sham Adx, kept on Zn- or Zn+ diets, were studied for ability to form
*Normal 0 Zn-
=in Ml-
Lab
Chow
01 0
1 2
I 4
6
8
IO
I 6 12
8 I4
Perio (weeks) Age (weeks)
FIG. 2. Increase in the number of autologous rosette-forming cells in the spleen of C57BVKs mice fed Zn-, Zn+, and normal lab chow diets for 3, 6, and 8 weeks. MRBC/Sc ratios at 64/l (mean f SD).
241
SHORT COMMUNICATIONS TABLE
1
Autologous Rosette-Forming Cells in the Spleen of C57BlIKs Mice, Adrenalectomized or Sham Adrenalectomized, Kept on Zn-, Zn+, and Normal Lab Chow Diets Groups
Groups
A-Rosettes”
Zn-
40 + 2
Zn+
16 r 2
Lab chow
19 -c 3
Zn
68 z!I 4
Zn+
162 1
Lab chow
19 r 0.7
A-Rosettes”
3 weeks” ZnZnZn+ Zn+ Lab
Adx sham Adx Adx sham Adx chow
30 50 11 13 21
L 4 ? 2 _f 1 2 0.1 + 2
6 weeks* ZnZnZn+ Zn+ Lab
Adx sham Adx Adx sham Adx chow
45 64 9 12 17
r+_0.9 t 0.8 rt 0.5 k 1 k 1
” A-Rosette-forming cells per 1000 spleen cells, mean 2 SD. b Mice maintained 3 and 6 weeks on the various diets were 11 and 14 weeks old, respectively, at time of analysis. They were Adx or sham Adx when 6 weeks old, fed a normal lab chow diet for 2 weeks, then placed on the Zn- or Zn+ diets for 3 and 6 weeks.
A-rosettes and compared to those of animals fed a normal lab chow diet. As shown in Table 1, spleen cells of mice Adx or sham Adx still formed high numbers of A-rosettes after 3 and 6 weeks on Zn- diet. The number of A-rosettes formed with spleen cells of Zn- Adx mice was lower than that of the Zn- sham-operated mice, but still much higher than was observed in mice fed Zn+ or lab chow diets. The inlluence of adrenalectomy or sham operation per se on the numbers of A-rosettes was insignificant in the mice fed Zn+ diet. In addition, spleen cells of mice kept 6 weeks on Zn- diet, then fed a normal lab chow diet for 2 weeks, showed marked decrease in their ability to form A-rosettes, as shown in Table 2. Serum zinc levels were measured in sera of each group of C57Bl/Ks mice which had been maintained on the different diets. As already described (9), mice on ZnTABLE
2
A-Rosette-Forming Cells in the Spleen of C57BllKs Mice Kept on Zn-, Zn+, and Lab Chow Diets for 6 Weeks and Then Fed a Normal Diet Containing Zn for 2 Weeks Groups” ZnZnZnZn+ Lab
Adx Adx + Zn+ diet sham Adx + Zn+ diet sham Adx chow
A-Rosettes* 45 17 25 13 21
+ s 4 2 rt
0.9 0.5 0.4 0.8 0.4
II Mice were 16 weeks old at time of study, those which were Zn- Adx were 14 weeks old. h A-Rosette-forming cells per 1000 spleen cells, mean 5 SD.
242
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diet had lower serum zinc level (40-53 &dl) than those fed Zn+ or lab chow diets (99-137 pg/dl). Animals kept on Zn- diet for 6 weeks, and subsequently fed the normal lab chow diet for 2 weeks, had normal serum zinc levels at time of study. DISCUSSION We have used the A-rosette marker as an approach to analyze the effect ofzinc on maturation of T lymphocytes, This marker appears to characterize a population of immature T cells which seem to be under thymic control, the number increasing after thymectomy (7). Our prior studies showed that zinc deficiency leads to a decrease in circulating. thymic factor, FTS (91, and produces defects of cellular function similar to those observed after thymectomy. Mice maintained on a Zn- diet for 3,6, and 8 weeks showed increasing number of cells in the spleen able to form A-rosettes as compared to mice fed normal or Zn+ diets administered by pair-feeding technique. The number of A-rosettes formed with spleen cells of Zn- Adx mice was lower than that of mice fed a Zn- diet, but still much higher than those fed Zn+ diet or lab chow diet. However, mice kept on a Zndiet for 6 weeks and then placed back on a normal diet containing zinc showed rapid return of the A-rosettes to the lower number that characterizes intact animals. Large numbers of cells able to form A-rosettes are present in thymus, spleen, and peripheral lymphoid tissues of thymectomized mice (7). But, under the influence of FTS, which remained at higher levels in normal mice or animals fed a Zn+ diet (9), low numbers of A-RFC were found, as expected. The increase of A-RFC in the spleen of Zn- mice may be attributable to an alteration in thymic function, decreasing FTS levels and permitting incomplete differentiation of T lymphocytes which could accumulate in peripheral lymphoid organs, i.e., spleen. Or this effect could be due to a loss of suppressor cells or to abnormal expressions of membrane antigens, i.e., Ly 1,2,3+ cells (4). Otherin viva studies (4,6) have demonstrated that the high level of A-RFC found after thymectomy could be decreased to normal levels following FTS injections. These findings support the view that T-cell maturation is associated with thymus function contributing to self-recognition and that loss of distinguishing self from nonself may have occurred in mice on Zn- diet. In the present study, we found the number of A-RFC to increase progressively when depletion of bodily Zn is produced by dietary restriction of this element. These findings are concordant with earlier studies in cattle suffering from the A-46 lethal mutation and in man with acrodermatitis enteropathica. In these diseases, Zn deficiency is consequent to failure of the normal absorption of Zn from the gastrointestinal tract, failure of normal thymus development, and profound deficiencies in immunity function (9, 12). These abnormalities are completely correctable when sufficient amounts of Zn are provided. Similarly, mice and rats rendered Zn deficient by dietary restriction of Zn show dramatic thymic involution and deficiency of cellularity in the T-dependent zones of lymph nodes and spleen (13), and administration of Zn promptly corrects the defects. It will be of great interest to determine whether Zn deficiencies in animals are associated with other evidence of immaturity among the peripheral lymphoid cells. Such markers as peanut agglutinin and terminal deoxyribonucleotidyl transferase should be studied. Since deficiency of zinc occurs frequently in humans as well, especially in association with immunodeficiencies and cancer (14,15), it will be of interest to see
SHORT COMMUNICATIONS
whether the A-rosette technique and, perhaps, other markers of immaturity cells, signal the Zn deficiency or can be correlated to it.
243 of T
ACKNOWLEDGMENTS We are most grateful to Dr. C. J. Menendez-Botet for the determinations of zinc levels in sera of C56BllKs mice under study. The skilled technical assistance of Ms. Pamela De Riso and Ms. Anne Manice are deeply appreciated. We are also grateful to Ms. Waleeratana Buthrasup for her kind assistance. This study was supported by USPHS Grants: CA-08748 CA-19267, CA-17404, CA-24476, AI-11843, NS-11457, and AG-00541, 3. M. Foundation, the Special Projects Committee of Memorial Sloan-Kettering Cancer Center, the Richard Molin Foundation, and the Ortho Pharmaceutical Corporation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15.
Good, R. A., “The Harvey Lectures,” Series 67. Academic Press, New York, 1973. Stutman, O., and Good, R. A., Transpl. hoc. 3, 923, 1971. Kadish, J., and Basch, R. S., J. Exp. Med. 143, 1082, 1976. Bach, J. F., Bach, M. A., Blanot, D., Bricas, E., Charreire. J., Dardenne, M., Fournier, C., and Pleau, J. M., Bull. Inst. Pasteur 76, 325, 1978. Charreire, J., and Bach, J. F., In “Biological Activity of Thymic Hormones” (D. W. van Bekkum, Ed.), pp. 245-250. Halstead Press Div., Wiley, New York, 1975. Charreire, J., and Bach, J. F., Proc. Nat. Acad. Sci. USA 72, 3201, 1975. Charreire, J., and Bach, J. F., Lancer 17, 229, 1974. Halstead, J. A., Smith, J. C., Jr., and Irwin, M. I., J. Nutr. 104, 347, 1974. Iwata, T., Cell. Immunol. 47, 100, 1979. “Analytical Methods for Atomic Absorption Spectrophotometry.” Perkin-Elmer, Norwalk, Conn., 1970. Luecke, R. W., Olman, M. E., and Baltzer, B. V., J. Nutr. 94, 344, 1968. Julius, R., Schulkind, M., Sprinkle, T., and Rennert, O., J. Pediutr. 83, 1007, 1973. Tanaka, T., Fernandes, G., Tsao, C., Pih, K., and Good, R. A., Fed. Proc. 37, 931, 1978. Cunningham-Rundles, C., Cunningham-Rundles, S., Garafolo, J., Iwata, T., Incefy, G., Twomey, J., and Good, R. A., Fed. Proc. 38, 1222, 1979. Davies, I. J., Musa, M., and Dormandy, T. L., J. Ch. P&ho/. 21, 359, 1968.
IMMUNOLOGY
48, 238-243
(1979)
Effect of Zinc Deficiency LEONARD
Memorial
NASH,
on Autologous
Rosette-Forming
TSUTOMLJ IWATA, GABRIEL FERNANDES, AND GENEVIEVE S. INCEFY’
Sloan-Kettering
Cancer
Center, Received
1275 York Avenue, April
New
ROBERT
York,
New
Cells A. GOOD,
York
10021
25, 1979
Ability to form autologous rosettes (A-rosettes) is characteristic of a certain population of immature T cells, present in the thymus of various species including man. In mice, few A-rosettes are normally found in peripheral blood and spleen but their number increases markedly in spleen following thymectomy. In our studies, A-rosette formation could be demonstrated to be significantly enhanced in the spleen of C57Bl/Ks mice after the animals had been maintained 3,6, and 8 weeks on a zinc-deficient diet (Zn-) and to increase progressively with duration of feeding the Zn diet. These changes were quickly reversed by feeding a normal diet containing zinc but could not be eliminated by complete adrenalectomy, a finding that ruled out their dependence on pituitary-adrenal as is function attributable to stress. Pair-fed controls and mice fed a zinc-supplemented diet ad l&turn showed few A-rosettes, as expected.
INTRODUCTION The maturation of lymphoid stem cells into peripheral T lymphocytes involves several stages of development. An initial step appears to depend on direct interaction with epithelium in vitro during passage of precursors through the thymus while subsequent stages of differentiation are attributable to influence of thymic hormones or circulating serum thymic factors (l-4). Ability to form autologous rosettes (A-rosettes) is characteristic of a certain population of immature T cells, which have been demonstrated in the thymus of various species, including man (5, 6). In mice, few A-rosettes are normally present in peripheral blood and spleen, but such cells are markedly increased in the spleen following thymectomy (7). Treatment with thymic hormone in vitro reduces the number of A-rosettes in the blood and spleen of thymectomized mice (7). Zinc, an essential metal for maximum activity of many enzymes (8), has also been demonstrated to be necessary for the development and maintenance of the thymus and for providing normal levels of circulating serum thymic factor, “facteur thymique serique” (FTS) (9). It was of interest, therefore, to study what influence Zn deficiency might have on such a population of precursor or immature T cells, and to attempt to correlate changes in number of A-rosette-forming cells (A-RFC) with the severity of Zn deficiency. A-rosette formation could be demonstrated to be enhanced in the spleen of C57Bl/Ks mice after the animals had been maintained 3 weeks on a Zn-deficient ’ To whom correspondence
and reprint requests should be addressed.
000%8749/79/130238-06$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
239
diet (Zn-), and more pronounced increases of A-rosettes were observed after 6 and 8 weeks on this diet. Changes in this immature T-cell population corresponded with the fall of FTS to low levels during Zn restriction. These changes were quickly reversed by feeding a normal diet containing zinc but could not be eliminated by complete adrenalectomy, a finding that ruled out their dependence on pituitaryadrenal as is function attributable to stress. Pair-fed controls and mice fed a Zn-supplemented diet (Zn’) ad libitum showed few A-rosettes, as expected. MATERIAL
AND METHODS
Animals and diets. Male and female C57Bl/Ks mice were divided into seven groups when they were 6 weeks old. Thirty-five mice were fed Purina lab chow ad libitum as controls, 50 were fed a zinc-deficient (Zn-) diet, and 50 a zinc-supplemented (Zn+) diet (Taklad test diets). Further, the Zn- and Zn+ groups were divided into adrenalectomized (Adx) and sham Adx groups of 10 mice each. The zinc content of the Purina lab chow was analyzed by atomic absorption spectrophotometry (10) and found to be 9.68 pg Zn/g. The 50 kg Zn/g were present in the Zn+ diet which was made up of egg white, dextrose, corn oil, and cellulose with vitamin and mineral supplements as described by Luecke et al. (11). No zinc was present in the Zn- diet and its composition was otherwise identical to the Zn+ diet. During the period of the study, mice were kept in stainless-steel cages, as previously described (9). They were fed ad fibitum and had free access to deionized distilled water, but had no contact with shavings or other material that could possibly contain zinc. Autologous rosette assay. The procedure utilized for A-rosette formation has been described in detail by Charreire and Bach (5, 6). In brief, it consisted of combining autologous or syngenic mouse erythrocytes (MRBC) and spleen cells (SC) in various MRBC/Sc ratios (16/l to 128/l). Scoring of rosettes was performed as previously described (5) except in certain cases (64/l, 128/l) when 1 and 1.25 ml of medium were added, respectively, to dilute the cell suspensions permitting easier reading. Adherent cells were depleted by prior incubation of the spleen cells for 90 min at 37°C before adding the MRBC. RESULTS Spontaneous A-rosette formation of spleen cells with mouse erythrocytes was analyzed at different MRBC/Sc ratios. Initially, spleen cells of C57BV6 Cum mice, 12 weeks old, that had been thymectomized 4 weeks earlier, were tested for spontaneous A-rosette formation. The results are shown in Fig. la. This analysis established the desired ratios of MRBC/Sc to be used for demonstration of A-RFC in mice kept on Zn- and Zn+ diets. These procedures were then applied to the analysis of spleen cells of C57Bl/Ks mice, 9 and 14 weeks old, that had been maintained for 3 and 8 weeks on Zn- diet to verify the optimal MRBC/Sc ratio at which A-rosettes should be formed with cells of these animals. The results were compared to those obtained with spleen cells from lCweek-old normal C57BVKs mice (Fig. lb). The number of cells able to form A-rosettes in the spleen of animals fed Zn-, Zn+, and lab chow diets for 3,6, and 8 weeks were investigated using optimum ratios of MRBC/Sc of 64/l. A-RFC in spleen of mice maintained on Zn- diet had increased
240
SHORT COMMUNICATIONS
Ii : : & 01
I
I
I
16
32
64 Mouse
1 128 RBC/Spleen
16 Cells
32 (MRBC/W
I
I
64
128
FIG. 1. Number of autologous rosette-forming cells according to mouse erythrocytes/spleen cells ratios. (a) Spleen cells of 12-week-old C57BV6 Cum mice tested 4 weeks after thymectomy, (0); normal controls, (0). (b) Spleen cells of C57BYKs mice, 9 weeks old, fed 3 weeks the Zn- diet, (Cl); 14 weeks old, fed 8 weeks the Zn- diet, (A); 14 weeks old, fed normal lab chow diet, (A) (mean of two or three determinations f SD).
significantly (Fig. 2). It can be seen that A-RFC increased progressively with duration of feeding the Zn- diet. Few A-RFC were present in the spleens of animals fed lab chow ad libitum or Zn+ diets, using the pair-feeding method. The effects of stress caused by the zinc deficiency was also analyzed. Spleen cells of mice Adx or sham Adx, kept on Zn- or Zn+ diets, were studied for ability to form
*Normal 0 Zn-
=in Ml-
Lab
Chow
01 0
1 2
I 4
6
8
IO
I 6 12
8 I4
Perio (weeks) Age (weeks)
FIG. 2. Increase in the number of autologous rosette-forming cells in the spleen of C57BVKs mice fed Zn-, Zn+, and normal lab chow diets for 3, 6, and 8 weeks. MRBC/Sc ratios at 64/l (mean f SD).
241
SHORT COMMUNICATIONS TABLE
1
Autologous Rosette-Forming Cells in the Spleen of C57BlIKs Mice, Adrenalectomized or Sham Adrenalectomized, Kept on Zn-, Zn+, and Normal Lab Chow Diets Groups
Groups
A-Rosettes”
Zn-
40 + 2
Zn+
16 r 2
Lab chow
19 -c 3
Zn
68 z!I 4
Zn+
162 1
Lab chow
19 r 0.7
A-Rosettes”
3 weeks” ZnZnZn+ Zn+ Lab
Adx sham Adx Adx sham Adx chow
30 50 11 13 21
L 4 ? 2 _f 1 2 0.1 + 2
6 weeks* ZnZnZn+ Zn+ Lab
Adx sham Adx Adx sham Adx chow
45 64 9 12 17
r+_0.9 t 0.8 rt 0.5 k 1 k 1
” A-Rosette-forming cells per 1000 spleen cells, mean 2 SD. b Mice maintained 3 and 6 weeks on the various diets were 11 and 14 weeks old, respectively, at time of analysis. They were Adx or sham Adx when 6 weeks old, fed a normal lab chow diet for 2 weeks, then placed on the Zn- or Zn+ diets for 3 and 6 weeks.
A-rosettes and compared to those of animals fed a normal lab chow diet. As shown in Table 1, spleen cells of mice Adx or sham Adx still formed high numbers of A-rosettes after 3 and 6 weeks on Zn- diet. The number of A-rosettes formed with spleen cells of Zn- Adx mice was lower than that of the Zn- sham-operated mice, but still much higher than was observed in mice fed Zn+ or lab chow diets. The inlluence of adrenalectomy or sham operation per se on the numbers of A-rosettes was insignificant in the mice fed Zn+ diet. In addition, spleen cells of mice kept 6 weeks on Zn- diet, then fed a normal lab chow diet for 2 weeks, showed marked decrease in their ability to form A-rosettes, as shown in Table 2. Serum zinc levels were measured in sera of each group of C57Bl/Ks mice which had been maintained on the different diets. As already described (9), mice on ZnTABLE
2
A-Rosette-Forming Cells in the Spleen of C57BllKs Mice Kept on Zn-, Zn+, and Lab Chow Diets for 6 Weeks and Then Fed a Normal Diet Containing Zn for 2 Weeks Groups” ZnZnZnZn+ Lab
Adx Adx + Zn+ diet sham Adx + Zn+ diet sham Adx chow
A-Rosettes* 45 17 25 13 21
+ s 4 2 rt
0.9 0.5 0.4 0.8 0.4
II Mice were 16 weeks old at time of study, those which were Zn- Adx were 14 weeks old. h A-Rosette-forming cells per 1000 spleen cells, mean 5 SD.
242
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COMMUNICATIONS
diet had lower serum zinc level (40-53 &dl) than those fed Zn+ or lab chow diets (99-137 pg/dl). Animals kept on Zn- diet for 6 weeks, and subsequently fed the normal lab chow diet for 2 weeks, had normal serum zinc levels at time of study. DISCUSSION We have used the A-rosette marker as an approach to analyze the effect ofzinc on maturation of T lymphocytes, This marker appears to characterize a population of immature T cells which seem to be under thymic control, the number increasing after thymectomy (7). Our prior studies showed that zinc deficiency leads to a decrease in circulating. thymic factor, FTS (91, and produces defects of cellular function similar to those observed after thymectomy. Mice maintained on a Zn- diet for 3,6, and 8 weeks showed increasing number of cells in the spleen able to form A-rosettes as compared to mice fed normal or Zn+ diets administered by pair-feeding technique. The number of A-rosettes formed with spleen cells of Zn- Adx mice was lower than that of mice fed a Zn- diet, but still much higher than those fed Zn+ diet or lab chow diet. However, mice kept on a Zndiet for 6 weeks and then placed back on a normal diet containing zinc showed rapid return of the A-rosettes to the lower number that characterizes intact animals. Large numbers of cells able to form A-rosettes are present in thymus, spleen, and peripheral lymphoid tissues of thymectomized mice (7). But, under the influence of FTS, which remained at higher levels in normal mice or animals fed a Zn+ diet (9), low numbers of A-RFC were found, as expected. The increase of A-RFC in the spleen of Zn- mice may be attributable to an alteration in thymic function, decreasing FTS levels and permitting incomplete differentiation of T lymphocytes which could accumulate in peripheral lymphoid organs, i.e., spleen. Or this effect could be due to a loss of suppressor cells or to abnormal expressions of membrane antigens, i.e., Ly 1,2,3+ cells (4). Otherin viva studies (4,6) have demonstrated that the high level of A-RFC found after thymectomy could be decreased to normal levels following FTS injections. These findings support the view that T-cell maturation is associated with thymus function contributing to self-recognition and that loss of distinguishing self from nonself may have occurred in mice on Zn- diet. In the present study, we found the number of A-RFC to increase progressively when depletion of bodily Zn is produced by dietary restriction of this element. These findings are concordant with earlier studies in cattle suffering from the A-46 lethal mutation and in man with acrodermatitis enteropathica. In these diseases, Zn deficiency is consequent to failure of the normal absorption of Zn from the gastrointestinal tract, failure of normal thymus development, and profound deficiencies in immunity function (9, 12). These abnormalities are completely correctable when sufficient amounts of Zn are provided. Similarly, mice and rats rendered Zn deficient by dietary restriction of Zn show dramatic thymic involution and deficiency of cellularity in the T-dependent zones of lymph nodes and spleen (13), and administration of Zn promptly corrects the defects. It will be of great interest to determine whether Zn deficiencies in animals are associated with other evidence of immaturity among the peripheral lymphoid cells. Such markers as peanut agglutinin and terminal deoxyribonucleotidyl transferase should be studied. Since deficiency of zinc occurs frequently in humans as well, especially in association with immunodeficiencies and cancer (14,15), it will be of interest to see
SHORT COMMUNICATIONS
whether the A-rosette technique and, perhaps, other markers of immaturity cells, signal the Zn deficiency or can be correlated to it.
243 of T
ACKNOWLEDGMENTS We are most grateful to Dr. C. J. Menendez-Botet for the determinations of zinc levels in sera of C56BllKs mice under study. The skilled technical assistance of Ms. Pamela De Riso and Ms. Anne Manice are deeply appreciated. We are also grateful to Ms. Waleeratana Buthrasup for her kind assistance. This study was supported by USPHS Grants: CA-08748 CA-19267, CA-17404, CA-24476, AI-11843, NS-11457, and AG-00541, 3. M. Foundation, the Special Projects Committee of Memorial Sloan-Kettering Cancer Center, the Richard Molin Foundation, and the Ortho Pharmaceutical Corporation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15.
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