Lymphocytes from copper-deficient mice exhibit decreased mitogen reactivity

NUTRITION RESEARCH, Vo]. 3, pp. 335-34], ]983 027]-53]7/83 $3.00 + .00 Printed in the USA. Copyright (c) ]983 Pergamon Press Ltd. A]] rights reserved.

LYMPHOCYTES FROM COPPER-DEFICIENT MICE EXHIBIT DECREASED MITOGEN REACTIVITY Omelan A. Lukasewycz and Joseph R. Prohaska Departments of Medical Microbiology & Immunology and Biochemistry, School of Medicine, University of Minnesota, Duluth; Duluth, MN 55812

ABSTRACT Copper deficiency was produced in C58 mice during perinatal development. Splenocytes from six-week old copper-deficient and copper-sufficient mice were evaluated for their ability to respond to various mitogens. When compared t o c e l l s from copper-supplemented control mice, splenic lymphocytes from copper-deficient animals showed a markedly decreased response to stimulation by the B cell mitogen, Lipopolysaccharide, and to the T cell mitogens Phytohemagglutinin and Concanavalin A, as well as to Pokeweed mitogen, a non-apeclficstimulator of both B and T cells. This decreased response was evident both in terms of [3H]-thymidine incorporation and in terms of stimulation index. The diminution of mitogenic reactivity to Concanavalin A was related to the degree of copper deficiency as e v a l u a t e d b y plasma cerul0plasmin activity. Chronic coppez deficiency results in an impairment of splenocyte T cell and B cell responsiveness. Key Words:

Copper-DeficientMice, Mitogen Responsiveness, B and T cell Mitogens. INTRODUCTION

The environment influences the generation and maintenance of the immune response. Within this context it is apparent that nutritional factors play major roles. The necessity of adequate trace metal nutriture for a proper i ~ u n e response is now firmly established (1-3). Recently, copper has been identified as a trace metal that influences the immune system (4). It has been shown that in vitro fungicidal activity is depressed in neutrophils from copper-deficient (-Cu) cattle (5) and sheep (6). Copperdeficient rats have a decreased resistance to infection with Salmonella typhimurium (7) and -Cu mice had a lower LDs0 to challenge by Pasturella hemolytica (D. G. Jones and N. F. Suttle, personal communication) than do the respective copper-sufficient controls. Our laboratory has developed a model system to evaluate the immune response of the -Cu mouse. Copper-deficient animals exhibit a decreased humoral immune response which appears to be proportional to the degree of copper depletion as reflected by serum ceruloplasmin levels (4). Cell mediated immunity, as measured by the ability of animals to be immunized to a syngeneic lymphocytic leukemia, is also much reduced or absent in -Cu animals (8). Flow cytometry studies utilizing a fluorescence activated cell sorter have indicated an alteration in lymphocyt e subpopulations in -Cu mice (9) characterized by a marked increase in B cells and a concomitant decrease in the T cell subpopulation,

335

336

O.A. Lukasewycz and J.R. Prohaska

mainly within the T-helper subset. Our present endeavor was to determine the extent to which B and/or T cells were compromised immunologically by an analysis of the mitogen reactivity of lymphocytes from -Cu mice compared to copper-supplemented (+Cu) controls. METHODS Mice. Inbred C58 mice, originally from Dr. William H. Murphy, Jr., Department of Microbiology, University of Michigan Medical School, Ann Arbor, have been maintained at the animal facilities of the School of Medicine of the University of Minnesota, Duluth, since 1975. Pregnant dams were housed in polycarbonate cages with stainless-steel covers and wood shavings for bedding. Offspring (usually no more than 46 per dam for C58 mice) were weaned when 3 weeks old and were transferred to stainless-steel cages with wire mesh floors at 4 weeks of age. Mice were used for mitogen reactivity experiments at 5 to 7 weeks of age. Diet. On the day of parturition C58 dams were switched from a non-purified diet (Purina Mouse Chow, Ralston Purina Co., St. Louis, MO) containing 1214 mg of copper per kilogram to a purified diet formulated to omit copper from the salt mix (modified AIN 76A, Teklad Laboratories, Inc., Madison, WI) which contained 0.6 mg copper per kilogram. This diet has previously been used to establish copper deficiency in mice (i0). Approximately half the dams were given supplemental copper (20 wg/ml as CuSO~) in their drinking water (+Cu), while the remaining dams were given deionized water to drink. The amount of copper available to the +Cu mice in terms of water volume consumed per day would be approximately equivalent to the amount of copper in "mouse chow" consumed per day. Pups were maintained on the treatment of their respective dams until the experiments began. For any given experiment, copper-supplemented (+Cu) and copper-deficient (-Cu) animals were matched as to age and sex and were of equivalent weight. -Cu animals not attaining the weight of their +Cu counterparts were discarded. Ceruloplasmin levels. Plasma from individual microhematocrit tubes was assayed for ceruloplasmin (EC 1.16.3.1) activity with o-dianisidine as substrate as described by Prohaska (i0). Cerul0plasmin, a cuproenzyme, was routinely used to monitor the copper status of the mice. Lymphocyte preparation. Fresh suspensions of splenocytes were prepared from experimentally matched +Cu and -Cu C58 mice. Spleens were removed aseptically from the appropriate donors, placed in Hanks Buffered Salt Solution (HBSS) and teased with scalpels to prepare a single cell suspension. Gross particles were allowed to settle, and the cell suspension transferred into a sterile tube, and centrifuged at 250 x g for i0 minutes. Cells were resuspended in media, counted with a hemocytometer, and diluted to a final concentration of 5 x 106 cells/ml. Viability of splenocytes was determined by trypan blue exclusion and was routinely greater than 90%. Media and Mitogen preparation. Unless otherwise indicated, the media used in these experiments was Dulbeeco's minimum essential medium (Gibco, Grand Island, NY) supplemented with 10mM HEPES, penicillin G (i unit/ml media), Streptomycin (i ~g/ml media), 2-mercaptoethanol (5 x 10-SM), imM sodium pyruvat~ 0.015 mM folic acid, 0.25 mM L-asparagine, 0.55 mM arginine, 150 mM L-glutamine and 0.5% autologous serum. Media was prepared fresh daily in i00 ml quantities. Final media contained 0.047 wg/ml of total copper as determined b y atomic absorption spectroscopy. Stock solutions of Concanavalln A (Con A, Sigma Chemical Company, St. Louis, MO), Lipopolysaccharide B (LPS) from E. eoli 0127:B8 (Difco Laboratories, Detroit, MI), Phytohemagglutinin P (PHA, Difco Laboratories, Detroit, MI) and Pokeweed Mitogen (PWM, Sigma Chemical Co., St. Louis, MO) were prepared in distilled water, dispensed in 0.5 ml quantities, and kept frozen at -20~ Samples were diluted to appropriate concentrations in media prior to use in the mitogen assays.

Mitogen Response in Cu Deficiency

337

Mitogen response. Spleen cell preparations, 5 x l0 s cells/0.1 ml media, were ~ s e d into 96-well microtiter plates (Gibco Laboratories, Grand Island, NY). Each well received an additional 0.1 ml of media containing the specified quantity of the appropriate mitogen. Each analysis was done in triplicate. One ~Ci (0.53 nmoles) o f [ 3 H ] - t h y m i d i n e (Schwartz-Mann, Orangeburg, NY) was added to each well for the last 18 hours of the 48 hour incubation (37~ 5% C02) period. Background incorporation was determined in wells that received additional media w i t h o u t m i t o g e n s . Cells were harvested with a multiple microharvester (Otto Hiller Co., Madison, WI) and collected on strips of glass fiber filters (Whatman Co., Clifton, NJ). Filters were dried with infrared heat lamps, placed in v i a l s w i t h 5 ml of scintillation cocktail (containing PPO:POPOP:toluene at 4.0 gm:0.1 gm:l liter respectively) and counted in a Beckman LS 315OT scintillation counter. Results of triplicate samples were expressed as mean cpm • S.D. A stimulation index (S.I.) for mitogen reactivity analyses was determined for some experiments by the formula: S.I. = cpm of cell culture at the optimal dose of mitogen cpm of cell culture without mitogen Statistics. Mean differences were compared by either Student's t-test or by analysis of variance (randomized complete block design with subsampling) using the F-test at two levels of ~, 0.05 and 0.01 (ii). Population variances were tested for equality using the F-test. RESULTS Mice used for the splenocyte culture studies were nursed by dams fed the -Cu diet with or without supplemental copper a n d were themselves continued on the respective treatment following weaning. The -Cu mice had low levels of copper in their livers as indicated by plasma ceruloplasmin activities which averaged 2.79 • 1.9 units/liter compared to values of 13.2 • 1.9 units/ liter for the +Cu mice. The -Cu mice also had functional deficits of copper in lymphoid tissue since, compared to -Cu mice, the activities of two cuproenzymes, cytochrome oxidase and superoxide dismutase, were lower in spleen and thymus of -Cu animals (J. R. Prohaska and O. A. Lukasewycz, unpublished results). A total of 92 mice, 52 +Cu and 40 -Cu, were tested for their mitogenic responsiveness in splenocyte cultures. A typical set of dose-response curves of +Cu and -Cu splenocyte preparations to activation by the various B and T cell mitogens is presented in Figure i. Optimum responses of the +Cu splen0cytes was determined to be 0.5 ~g/ml for Con A; 5.0 ~g/ml PHA; 500 ~g/ml LPS; and 50 ~mg/ml PWM. The -Cu splenocyte response peaked at approximately these same concentrations but at a response level that was less than one-half of the +Cu values for all mitogens tested. The base line levels ([3H]-thymidine incorporation with no mitogen in the wells) were often higher in -Cu splenocytes than in +Cu preparations. For example, the base line levels for the experiments portrayed in Figure 1 were: +Cu = 1,865 • 287, -Cu = 3,314 • 694. Differences between the +Cu and -Cu dose response were significant, P < 0.01. Determination of stimulation indices for many such experiments (Fig. 2) more succinctly described the diminution in responsiveness of the -Cu splenocytes to the mitogens. While the overall stimulation index appears quite variable, the response of -Cu lymphocytes was always lower than that of +Cu cells within individual experiments. The meanstimulation indices • S.D. (Fig. 2) were as follows: Con A: +Cu = 57.5 • 18.8, -Cu = 12.9 • 11.3; PHA: +Cu = 23.8 • 13.9, -Cu = 11.9 • 12.5; LPS: +Cu = 19.6 • 6.9, -Cu = 5.7 • 6.4; and PWM: +Cu = 23.0 • 6.7, -Cu = 10.5 • 7.7. Thus, the response of -Cu C58 lymphocytes was less than one-half of the control value for each of the mitogens tested. These mean differences were statistically significant in all cases

338

O.A. Lukasewycz and J,R. Prohaska

ConA

100

60

80

40

60

80

40

2O 10

20 "4 0

2 x

o

eO

0.01

0.1

0,5

FIG. 1

1.0

0.1

1.0

10,0

20

10o

~'VM

50

40

=o[

30

LP8

20 slk~

10

10

I

10

100

0

2

Reactivity of copper-deficlent (o) and copper-supplemented (s) mice to the mitogens Concanavalin A (Con A), Phytohemegglutinin (PHA), Pokeweed mitogen (PWM) and Lipopolysaccharide (LPS). The mitogen response of splenocytes was determined in triplicate for each of the indicated dOses of the designated mitogens. IncorpOration of [3H]-thymidine was determined and the mean • S.D. presented.

Dose ~glwel)

PHA

ConA ,,x, 80

LPS

PWM

50

100

e

~e

40 o

60

30

|9

9e

-J

:D

4o

|0

o

20 ~

I

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o

oo 9

20

0

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o

9

o

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o

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o9 o9

10 9

O

9

o

o~

~o

..............

Cu +

Cu-

C/;~" Cu-

c.+

cu-

'Cu+' cu-

FIG. 2 S t i m u l a t i o n i n d i c e s o f s p l e n o c y t e s from c O p p e r . s u p p l e m e n t e d ( + C u ) ( s ) and copper-deficlent (-Cu) (o) mice to mitogen activation. Stimulation index was determined at optimal dose of mitogen.

Mi togen Response i n Cu Deficiency

339

(Con A, LPS, PWM, P < 0.01 and PHA, P < 0.05). Decreased mit0gen reactivity has a l s o b e e n observed in splenocytes f r o m - C u C57BL mice (0. A. Lukasewycz and J. R. Prohaska, unpublished results). To determine if the level of cerul0plasmin (i.e., degree of copper deficit) might be correlated to the amplitude of the mit0genie response, four experiments were selected where +Cu[SH]-thymidlne incorporation values were approximately equivalent and where -Cu ceruloplasmin levels were variable. Each splenocyte culture was obtained from a pool of mice~ The degree of stimulation of -Cu splenocytes by Con A appeared to decrease in -Cu m i e e w i t h decreasing serum cerul0plasminvalues (Table i). Even -Cu m i c e w i t h cerul0plasmin levels 80% of normal had reduced responsiveness to Con A (Table i).

TABLE 1 Mitogenic Capacity of Concanavalin A

Splenocyte source

Ceruloplasmin (units/liter)

SH-thymidine incorporationa . (cpm/well)

1-4

+Cu

19.2 • 2.35

173,941 • 8,531 b

1

-Cu

15.4

81,438 • 10,180 c

2

-Cu

6.5

20,622 • i0,916 c

3

-Cu

0.4

13,145 • 2,256 c

4

-Cu

0.4

9,264 • 3,087 c

Exp. No.

aAliquots of 5 x l0 s cells in a final volume of 0.2 ml media were incubated in 96 well microtlter plates with 0.i ~g/well Concanavalin A. One ~Ci of [SH]-thymidine was introduced for the last 18 hrs of the 48 hr culture. Cells were harvested and counted in a scintillation counter. Six wells were run for each experiment, values are means • S.D. bA m i n i m u m o f three mice were pooled for each individual experiment. The +Cu Values are means • S.D. of all four of the experiments. Csignificantly different from +Cu Value (P < 0.01).

DISCUSSION The capacity of splenocytes to mount a blastogenic response to mitogen stimulation is much diminished in -Cu animals compared to their +Cu counterparts. As shown in the dose-response curveS, individual -Cu m i c e s h o w a decreased response to the T cell mit0gens Con A and PHA and to the B cell mitogen LPS. The response to PWM, which causes blast transformation of both B and T cells, is also significantlydiminished. These observations are further substantiated b y the analysis ofstimulatlon indices. While the response to the indicated mitogens is subject to some variability~ the overall diminished response of -Cu mice is quite evident in each case. Within individual experi-

340

O.A. Lukasewycz and J.R. Prohaska

ments, the response of -Cu animals has never exceeded that observed in +Cu mice. The data presented herein thus strongly suggest that dietary copper deficiency in C58 mice leads to a diminished responsiveness on the part of both B and T lymphocytes. Furthermore, there is a strong suggestion, at least ir~ terms of the Con A response, that this decreased responsiveness is dependent upon the degree of copper deficiency as reflected by serum ceruloplasmin activity. This observation islanalogous to one in similar studies reported previously, namely, that the diminished antibody response to sheep red blood cells was proportional to the level of serum ceruloplasmin in -Cu mice (4). Thus, it would appear that both cell mediated immunity and humoral immunity are lower i n c o p p e r deficiency and in proportion to the severity of the copper deficit. It is of interest to note that a "slight" decrease in serum ceruloplasmin levels leads to significant alterations in both antibody production (4) and in mitogen responsiveness. The immunological consequences of copper deficiency in humans may be significant since the typical American diet is marginally adequate in copper (12). It is clear that adequate copper nutriture is essential to elicit both a humoral and cell mediated immune response. The current in vitro studies support previous in vivo work (8) in -Cu C58 mice and clearly demonstrate a defect in cell mediated immunity in copper deficiency. The decreased immune response seen in this -Cu mouse model may explain the increased morbidity in -Cu mice and rats (7) following bacterial challenge and the frequent infections reported in children with Menkes' disease. It is as yet difficult to ascertain whether the decreased reactivity of -Cu splenocytes is indicative of a functional and/or maturational insufficiency. It is certainly possible that decreased activity of copper-dependent enzyme systems may lead to a diminished response. However, a slow-down or shift in the maturational scheme of the immunocytes may also be responsible. Concurrent studies in our laboratory (9) utilizing cytofluorometric analysis indicate that a major population shift occurs in the splenoeytes of -Cu mice. This alterationinCludes an increase of surfac~ immunoglobulin-positive (B) cells and a concomitant decrease in the Lyt 1-2-3- (Helper T) cell population. In the normal developmental scheme, the response to Con A and PHA is usually at maximum levels between 30 and 60 days of age (13,14). The response of splenocytes to PWM peaks earlier, b y day 25 (15). LPS stimulates a rather mature B cell population (16,17). It is possible that the -Cu splenocytes have not matured properly or in numbers sufficient to elicit the appropriate response. It is also quite possible that B cells and T cells are not affected simultaneously or to the same extent. Preliminary data from our laboratory indicate that the response to LPS is much less depressed than is the Con A response in mice that are moderately copper deficient. A longitudinal study would be useful to determine if a delayed maturation phenomena exists. ACKNOWLEDGEMENTS The skilled technical assistance of Karen Kolquist, Dean Cox and William Bailey is appreciated. This work was supported by grant HD 15941 from the National Institutes of Health and a University of Minnesota Graduate School Grant. REFERENCES i.

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Mitogen Response in Cu Deficiency

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5.

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6.

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7.

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8.

LUKASEWYCZ, O.A. and PROHASKA, J.R. Immunization against transplantable leukemia impaired in copper-deficient mice. J. Natl. Can. Inst. 69: 489-493, 1982.

9.

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STEEL, R.G.D. and TORRIE, J.H. McGraw-Hill, New York, 1960.

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13.

GOLDSTEIN, A.L., GUHA, A., HOWE, M.L. and WHITE, A. Ontogenesis of cellmediated immunity in murine thymocytes and spleen cells and its acceleration by ~hymosin, a thymic hormone. J. Immunol, 106:773-780, 1971.

14.

ADLER, W.J., TAKIGUCHI, T., HARSH, B. and SMITH, R.T. Cellular recognition by mouse lymphocytes in vitro: I. Definition of a new technique and results o f s t i m u l a t i o n b y phytohemagglutinin and specific antigens. J. Exp. Med. 131:1049-1078, 1970.

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HOWE, M.L. and MANZIELLO, B. Ontogenesis of the in vitro response of murine lymphoid cells to cellular antigens and phytomitogens. J. Immunol. 109:534-539, 1972.

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GRONOWICZ, E., COUTINHO, A. and MOLLER, G. Differentiation of B cells: sequential appearance of responsiveness to polyclonal activators. Scand. J. Im~un01. 3:413-421, 1974.

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FIDLER, J.M., MORGAN, E.L. and WEIGLE, W.O. B lymphocyte differentiation in the CBA/N mouse: a delay in maturation rather than a total arrest. J. l=~un01. 124:13-18, 1980.

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Am. J. Clin.