Alterations in Splenic Lymphoid Cell Subsets and ...

Nutrition and Immunology

Alterations in Splenic Lymphoid Cell Subsets and Activation Antigens in Copper-Deficient Rats1 SHCIKAL BALA,

MARK L FAILLA2 AND JOAN

K. LÃœNNEY*

Vitamin and Mineral Nutrition Laboratory, Beltsuille Human Nutrition Research Center, and *Helminthic Diseases Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsuille, Maryland 20705

MATERIALS AND METHODS Animal care, diet and experimental design. Unless otherwise indicated, first parity, pregnant (13-15 d) Lewis rats were purchased from Charles River Labora tories (Wilmington, MA). Animals were maintained and handled in accordance with the guidelines of the U.S. Public Health Service and the U.S. Department of Agriculture. Details have been described elsewhere (9). Pregnant rats were fed a semipurified diet con-

INDEXING KEY WORDS:

â€¢copper â€¢lymphocytes â€¢lnterleukln-2 receptor â€¢transferrin receptor â€¢rats

The micronutrient Cu is required for numerous biological processes including immunocompetence (1). Severe Cu deficiency impairs both the innate (2, 3) and the acquired (4-9) branches of the immune system. The cell types that participate in the acquired immune responses include antigen presenting cells (APC)3, T-lymphocytes and B-lymphocytes. Whether inadequate Cu intake directly influences the matu

'This work was presented in part at the 74th Annual Meeting of the Federation of American Societies for Experimental Biology, Washington, DC, April 1990 [Bala, S., Pailla, M. L. &.Lunney, J. K. (1990) Chronic copper deficiency alters the cellularity, expression of E.-2 and transferrin receptors, and function of rat splenocytes. FASEBJ. 4: A1040 (abs. 4494)]. 2To whom correspondence should be addressed. Abbreviations used: APC, antigen presenting cells; Con A, Concanavalin A; IL-2R, interleukin-2 receptor; LPS, lipopolysaccharide; PHA, phytohemagglutinin; TfR, transferrin receptors.

0022-3166/91 $3.00 Â©1991 American Institute of Nutrition. Received 16 March 1990. Accepted 24 September 1990. 745

Downloaded from jn.nutrition.org at OHIO STATE UNIVERSITY on August 7, 2014

ration of accessory and effector immune cells or their ability to interact with one another is not known. Several recent studies have shown that the pheno typic profiles of the cells in mouse spleen (5, 10) and in rat peripheral blood (8, 9) are altered by dietary Cu deficiency. In this study we examined the effects of dietary Cu restriction on the phenotypic profile and mitogenic reactivity of rat splenocytes. The results show that both the severity of Cu deficiency and the sex of the animal influenced the impact of dietary Cu deficiency on these parameters. In vivo and in vitro expression of interleukin-2 receptor (IL-2R)and transferrin receptor (TfR) on splenic mononuclear cells also were assessed to determine if the impaired mitogenic response caused by Cu deficiency reflected a general attenu ation of events associated with cellular activation.

ABSTRACT Rats were nursed by dams fed a diet con taining adequate (6 |Â¿g/g)or deficient (0.6 ug/g) Cu during the lactation period and weaned to the same diet. Splenic mononuclear cells were isolated and the pheno typic profile determined by flow cytometry after Â¡mmunolabelling with monoclonal antibodies to cell surface markers. Total splenic mononuclear cell yield and the relative percentage and absolute number of T-cells and the CD4* (helper) and CDS* (cytotoxic) T-subsets were decreased in Cu-deficient male rats. The relative per centage, but not the absolute numbers, of splenic Bcells and macrophages was increased by Cu deficiency. The percentage of splenic mononuclear cells from male rats that expressed interleukin-2 receptors and transferrin receptors in vivo was increased by Cu deficiency. In contrast, dietary Cu deficiency did not affect the yield and phenotypic profile of splenic mononuclear cells in female rats. Reactivity of splenic mononuclear cells to Tcell mitogens was decreased in Cu-depleted male and female rats. However, mitogen-induced increases in levels of interleukin-2 receptor and transferrin receptor were similar in cultures of splenic mononuclear cells obtained from control rats and rats subjected to re stricted dietary intake of Cu only during the postlactation period. Thus, decreased mitogenic blastogenesis on ex posure of cells from Cu-deficient rats does not reflect a nonspecific impairment of cellular activation. J. Nutr. 121: 745-753, 1991.

746

BALA ET AL.

min) and filter sterilized prior to its use. Cell via bility, as determined by exclusion of trypan blue, exceeded 95%. The cell concentration was adjusted to 2 x IO6 viable cells/mL. Analysis of cell surface markers by flow cytometry. The procedure for immunolabelling of cell surface markers and subsequent analysis by flow cytometry has been described (9). The mouse antirat monoclonal antibodies used in this study were pur chased from Accurate Chemical Scientific (Westbury, NY) and included the following: W3/13 (T cells), W3/ 25 (CD4), OX8 (CD8), OX41 (macrophage/monocyte), OX39 (IL-2R) and OX26 (TfR). Fluorescein isothiocyanate (FITC(-labeled antimouse IgG (no cross-reactivity with rat IgG) and antirat IgG were

purchased from Organon Teknika (West Chester, PA). The flow cytometer was gated on viable mononuclear cells to exclude cell debris and erythrocytes. The percentage of fluorescent positive cells was calculated by subtracting integrated percent positive cells that were not treated with monoclonal antibodies (back ground) from integrated percent positive cells in test samples. Background values ranged from 3 to 5%. Fluorescent intensity of positively stained cells was also monitored. The relative intensity of staining, which is expressed as log mean channel, measures the average intensity of staining and indicates the molecular density of each antigenic determinant on the cell surface. Mitogenic reactivity in vitro. Aliquots (100 UL)of splenic mononuclear cells suspended in complete medium containing 2% homologous serum were added to 96-well plates. An additional aliquot (100 |1L) of either complete medium or complete medium with one of the following mitogens (Sigma Chemical, St. Louis, MO) was then added to each well: phytohemagglutin (PHA, 25 ug/well), concanavalin A (ConA), 1 ug/well), or lipopolysaccharide (LPS, 1 ug/ well). The indicated concentrations of mitogens were found to induce optimal reactivity in prehminary studies. Cultures were incubated for 72 h in 5% CO2: 95% air at 37'C. [3H]Thymidine [37 KBq (1 uCi); specific activity - 247.9 GBq (6.7 Ci)/mmol)] was added to each well 18 h before termination. Cultures were harvested onto glass-fiber filters using a semiautomated harvester and processed by standard proce dures. Results are expressed as A cpm between cells cultured in the presence and absence of each mitogen. This value was obtained by subtracting cpm [3H]thymidine incorporated in unstimulated cultures from cpm [3H]thymidine incorporated in cultures ex posed to test mitogens. Efficiency of counting for *H was 46-47%. All parameters were tested in triplicate with cells from each rat. Statistics. Data from male and female rats were separately subjected to ANOVA, and the StudentNewman-Keuls multiple range test was used to de termine significant differences (P < 0.05).

RESULTS Copper Status Male rats that had been nursed by dams fed a Cudeficient diet and weaned to this diet were charac terized by lower body weight, decreased levels of hepatic Cu and enlarged hearts (Table 1). We found that Cu-deficient male rats from one bitter had signifi cantly decreased hematocrits, larger spleens and livers and smaller thymuses than Cu-adequate controls. In contrast, hematocrits and relative sizes of these


taining sucrose (62.37 g/100 g), spray dried-egg white (20 g/lOOg),com oil (9.5 g/100 g), cellulose (3 g/100 g), AIN-76A mineral mix (3.5 g/100 g, 11), AIN-76 vi tamin mix (l g/100 g; 11), choline bitartrate (0.27 g/ 100 g) and biotin (0.2 mg/100 g). After parturition, dams were fed either the control (6 ug Cu/g) or the Cu-deficient (0.6 jig Cu/g) diet. To prepare the Cudeficient diet, cupric carbonate was not added to the mineral mix. Dams nursed 8-10 pups each. At 21 d of age, rats were weaned and fed the same diet as their mothers. For an experiment assessing the effect of Cu deficiency on the expression of IL-2R and TfR by splenic mononuclear cells in vitro, weaned male Lewis rats were purchased, housed and fed either Cudeficient or Cu-adequate diet as described here. At 8 wk of age, the rats were killed by decapitation, and truncal blood was collected for measurement of hematocrit and preparation of serum. Heart, thymus, spleen and liver were removed and weighed. Repre sentative pieces of liver, samples of diets and certified reference standards (bovine liver 1577a and rice flour 1568) from National Institute of Standards and Tech nology (Gaithersburg, MD) were digested and ana lyzed by atomic absorption spectrophotometry as described elsewhere (7). Recovery of Cu exceeded 95% as assessed by analysis of certified standards. Sera were directly analyzed by atomic absorption spectrophotometry after diluting 1:4 with deionized water. Isolation of mononuclear cells from spleen. Spleens were aseptically removed from rats, weighed and briefly stored in Hank's balanced salt solution at O'C before being washed and minced in the same solution. After removal of residual tissue using a nytex filter (37-nm pore size), the cellular suspension was underlayered with Histopaque (density 1.083 g/ mL) and centrifuged at 400 x g for 30 min at 22Â°C. Mononuclear cells were collected at the interface and washed three times with Hank's balanced salt solu tion. The final cell pellet was suspended in RPMI 1640 containing 2% pooled homologous serum that had been obtained from either Cu-deficient or control male rats. Serum was heat inactivated (56*C for 30

COPPER AND SPLENIC CELLULARITY AND FUNCTION

747

TABLE 1 Effect of dietary copper on body weights, hepatic copper, hematocrits and relative organ weights of mate rats' -Cu 13)218Body weight, gHepatic

ng/gHematocrit, Cu, PCV1Relative% weights,g/100 organ eightHeartSpleenThymusLiver+Cu(a g body vf

6a3.13Â± 0.22*44Â± 1"0.39Â±

0.01'0.25 Â± 0.01'0.28 Â± 0.01'4.64 Â± Â±0.19aGroup

B

AIn -5}182

-5)170 (a

3k0.77Â± 0.07"41Â± 1Â«0.82 Â±

&0.42Â± 0.09b28Â± 3k0.85Â±

0.02b0.30 Â± 0.03''b0.26 Â± 0.01'5.04 Â± Â±0.13a*Group

0.04b0.32 Â± 0.02"0.17 Â± 0.01b5.43 Â± Â±O.Kf"

organs in Cu-deficient male rats from a different litter were similar to controls. Moreover, the hepatic Cu content of rats with decreased hematocrit was only 13% that of the controls; Cu-deficient rats with normal hematocrits had hepatic Cu levels 25% that of the Cu-adequate controls. Consequently, Cu-defi cient rats were subdivided into two groups: group A (normal hematocrit) and group B (low hematocrit) to examine whether the apparent severity of Cu defi ciency influenced the phenotypic profile and mitogenie reactivity of splenic mononuclear cells. Differ ences due to litter variation cannot be excluded be cause of the limited sample size, i.e., only two dams were fed the Cu-deficient diet. The female rats fed Cu-deficient diet were also characterized by low hepatic Cu and enlarged hearts, but exhibited normal growth (Table 2). Although these female rats were littermates of the Cu-deficient male rats assigned to groups A and B, the hematocrit was similar for all female rats fed Cu-deficient and control diets. Similarly, relative weights of lymphoid tissues and liver in female rats were not influenced by dietary Cu. Thus, the characteristics of Cu-deficient female rats were similar to Cu-deficient males as signed to group A. Splenic Cellulartty Despite the 20-25% increase in relative splenic weight in Cu-deficient male rats, the yield of mononuclear cells per spleen collected at the in terface of the gradient was about one-half that for control rats (Table 3). In addition, the phenotypic profile of the mononuclear cell population was altered

in Cu-deficient male rats. The relative percentages (Fig. 1) and absolute numbers (Table 3) of T lympho cytes and CD4 (T helper) cells were lower in both groups of Cu-deficient male rats than in Cu-adequate controls. The percentage and absolute number of splenic CDS (T cytotoxic) cells was also decreased in

b b

W3/13W3/25 T-cell CD4

0X8 CDS

slg B-cell

0X41 M0

FIGURE 1 Dietary cu deficiency alters phenotypic profile of splenic mononuclear cells in male rats. Results (mean Â± SEM) are expressed as percentage of splenic mononuclear cell isolated from +Cu (solid bars), -Cu group A (open bars) and -Cu group B (hatched bars) rats exhibiting positive fluorescence above background control. Hematocrit and rel ative weights of spleen and thymus were similar in +Cu and -Cu group A rats, but significantly different in +Cu and -Cu group B rats (see Table 1). Significant differences (P < 0.05, Student-Newman-Keuls multiple-range test)) between treatment groups are indicated by presence of different letters above the error bars. There were 13 rats for the +Cu group and five each for A and B groups of -Cu rats.


'Results represent mean Â±SEM.The Cu-deficient (-Cu) male rats from one litter were assigned to either group A or B. Each litter was nursed by its mother, who was fed a -Cu diet following parturition; weaned pups were fed the same diet. There were three dams in the Cuadequate (+Cu) group. The presence of different letters as superscripts within a row indicates that groups are significantly different at P < 0.05, Student-Newman-Keuls multiple range test. 2PCV - packed cell volume.

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TABLE 2 Effect of dietary copper on body weights, hepatic copper, hematocrits,

and relative

organ weights

-Cu (a -6)

+Cu (n - 12) Body weight, g Hepatic Cu, ug/g Hematocrit, % PCV1 Relative organ weights,

g/100 g body weight Heart Spleen

Thymus Liver

161

of female rats'

Â±4'

3.56 Â±0.0944 Â±1'

Â±2' 0.82 Â±0.1711 Â±2' 43

0.41 0.27 0.32 4.60

0.78 0.25 0.31 5.12

Â±0.01' Â±0.02' Â±0.02' Â±0. lo-

Â±0.04b Â±0.01' Â±0.01' Â±0.09*

Cu-deficient male rats, although this difference was significant only for most severely deficient rats, i.e., group B. The relative percentages of splenic B-cells and macrophages were significantly higher in all Cudeficient male rats than in their Cu-adequate con trols. Although the absolute numbers of these cell types were also decreased in the spleens of Cu-defi cient rats, the differences were not significant. In marked contrast with the above results for cells from male rats, the yield and the relative percentages (Fig. 2) and absolute numbers (Table 3) of T-cells, CD4 cells, CDS cells, B-cells and macrophages present in splenic mononuclear cell fraction from female rats were not affected by dietary Cu defi ciency.

Expression of lnterleukin-2 and Transferrin Receptors Numerous molecular events, including the ex pression of IL-2R and TfR, precede antigen-mediated induction of DNA synthesis and blastogenesis by lymphocytes (12). We compared the expression of these two receptors in freshly isolated and cultured splenic mononuclear cells from control and Cu-deficient male rats in initial studies aimed at determining the biochemical basis for the impaired reactivity of these cells to ConA.

Blastogenic Reactivity Incorporation of [3H]thymidine into DNA by cul tures of splenic mononuclear cells in the absence of mitogens (background) was not affected by either di etary Cu or sex (Table 4). However, in vitro blasto genesis of splenic mononuclear cells from male rats exposed to the T-cell mitogens PHA and ConA was depressed 75-80% by dietary Cu deficiency. Reac tivity to LPS, a B-cell mitogen, was also significantly depressed in cultures of mononuclear cells from Cudeficient male rats. PHA-induced blastogenesis of mononuclear cells from Cu-deficient female rats was 29% that of control cells. While the blastogenic response of cells from Cudeficient female rats to ConA was -65% that of con trols, this difference was not statistically significant. Similarly, reactivity of mononuclear cells from female rats to LPS was not modified by the level of dietary Cu.

Â«Â«5Ã¼9)>

*-*~woa-â€¢p

or-OU604020r>

W3/13 T-cell

W3/25 CD4

0X8 CDS

slg B-cell

0X41 M0

FIGURE 2 Phenotypic profile of splenic mononuclear cells is not altered in Cu-deficient female rats. Results (mean Â±SEM) are expressed as percentage of cells from +Cu (solid bars) and -Cu (open bars), with positive fluorescence above background control. Significant differences [P > 0.05, Student-Newman-Keuls multiple range test) between treatment groups were not found. There were 12 rats in the +Cu and six rats in the -Cu group.


'Results represent mean Â±SEM.The Cu-dcficicnt (-Cu) female rats were from the same two litters as -Cu males assigned to groups A and B in Table 1. There were three dams in the Cu-adequate (+Cu) group. The presence of different letters as superscripts within a row indicates that values are significantly different at P < 0.05, Student-Newman-Keuls multiple range test. 2PCV - packed cell volume.

COPPER AND SPLENIC CELLULARITY AND FUNCTION

749

TABLE 3 Effect of dietary copper deficiency

on total number

of mononudear

cell types in male and female rat spleens'

Males -Cu typeMononuclearpan Cell

TCD4CDSB-cellMacrophages+Cu21.7

Females

Bcell number per10.6 x IF7 1.4Â»5.1 Â± 1.3A9.2 Â± 1.6Â»6.4 Â± 1.6*13.0 Â± 0.8A6.6 Â± 0.7Â»3.3 Â± 0.8'9.6 Â± 1.2Â»3.7 Â± 0.7A2.7 Â± 0.3Â»0.9 Â± Â±0.7"3.1 0.5k1.5 Â± 0.2Â»3.4 Â± 0.3A6.6 Â± 0.3'5.1 Â± 0.4'-Â»3.9 Â± 0.6*2.0 Â± 0.6A1.7 Â± 0.6'3.0 Â± 1.0-2.3 Â± Â±0.3'Group Â±0.4'Group Â±0.2'+Cuspleen16.8 Â±0.2A-Cu12.5 A12.4

1.6A6.9 Â± !.()*â€¢4.5 Â± 0.6A1.9 Â± 0.2A5.3 Â± 0.8A1.8 Â± Â±0.4A

Freshly isolated cells. The percentage of freshly isolated splenic mononuclear cells from male rats expressing IL-2R was increased by Cu deficiency in itiated during the lactation period (Fig. 3). Also, the relative intensity of staining, an indicator of receptor density on the cell surface, was significantly higher on mononuclear cells isolated from the more severely Cu-deficient male rats (group B = mean channel of log 3.9 Â±0.2) than on mononuclear cells from less se verely Cu-deficient rats (group A = mean channel of log 3.0 Â±0.1) and control animals (mean channel of

log 2.7 Â±0.1). In contrast, neither the percentage of splenic mononuclear cells expressing IL-2R (Fig. 3) nor the relative intensity of IL-2R in female rats was altered by dietary Cu deficiency. The percentage of freshly isolated splenic mononuclear cells expressing the TfR was about twofold higher in Cu-depleted group B male rats than in Cu-adequate controls (Fig. 3). Although the per centage of mononuclear cells from Cu-deficient animals with normal hematocrit (group A) that ex pressed TfR was about 1.4-fold higher than for cells

TABLE 4 Effect of dietary copper deficiency on in vitro blastogenesis of splenic T cells from male and female rats1

GroupMale+Cu-Cugroup xÃ•0-3242

Agroup BFemale+Cu-Cun11355126None1.6

0.4'1.5 Â±

14'16 Â±

28a44 Â±

0.6'1.4 Â± 0.2'0.9 Â±

12Â»16 Â± 11Â»116 Â±

18Â»59 Â± 22Â»161 Â±

0.4A1.4 Â± Â±0.5APHA75

1.0a0.8 Â± 0.3Â»1.0 Â± 0.2Â»3.8 Â±

26A33 Â± 43A103 Â± 0.9*3.8 Â± Â± 7"MitogenConAcpm Â±49ALPS6.6 Â±1.4A

'Results are expressed as mean Â±SEM.Hematocrit and relative weights of thymus and spleen of Cu-deficient male rats assigned to group A were similar to +Cu males, whereas these parameters were significantly different in Cu-deficient males assigned to group B (see Table 1). 2Number of rats used to isolate splenic mononuclear cells for each group. Data were analyzed separately by gender. Different letters as superscripts within a column indicates that values for -Cu and +Cu rats are significantly different at P < 0.05, Student-Newman-Keuls multiple range test. Abbreviations used: PHA, phytohemagglutinin; ConA, Concanavalin A; LPS, lipopolysaccharide.


'Results are expressed as means Â±SEM.Hematocrit and relative weights of thymus and spleen of Cu-deficient (-Cu) males assigned to group A were similar to control males receiving diets adequate in Cu (+Cu);-Cu males assigned to group B had lower hematocrit, smaller thymus and larger spleens than their controls (see Table 1).Data for each sex were analyzed separately to determine significant differences. Number of donor rats for splenic mononuclear cells from each group is same as mentioned in Figures 1 and 2. Absolute number of splenic mononuclear cell types was calculated from the relative percentage of fluorescent-positive cells and total mononuclear cell yield per spleen. The presence of different letters as superscripts within a row shows that numbers of indicated cell types from the spleen of -Cu and +Cu rats are significantly different (P < 0.05), Student-Newman-Keuls multiple range test.

BALA ET AL.

750

Male

Female

Male

Female

from controls, this difference was not statistically significant (P - 0.14). The relative intensity of fluo rescent staining for TfR was also increased on mononuclear cells from group B Cu-deficient rats (mean channel of log 5.7 Â±0.2) as compared with cells from group A Cu-deficient rats (mean channel of log 3.4 Â±0.4) and Cu-adequate controls (mean channel of log 2.8 Â±0.2). Expression of TfR was not affected by dietary Cu deficiency in female rats (Fig. 3). Cultured cells, in this experiment splenic mononuclear cells were isolated from different groups of male control and Cu-deficient rats. These animals were obtained as weanlings and fed either a Cu-ade quate or Cu-deficient diet for 5 wk. Rats fed the Cudeficient diet for 5 wk postweaning had lower levels of hepatic and serum Cu, lower hematocrits and larger hearts than control rats (data not shown). Be cause the relative weights of thymus, spleen and liver were similar in rats fed Cu-deficient and Cu-adequate diets, the Cu-deficient rats in this study were similar to Cu-deficient male rats that had been nursed by dams fed a Cu-deficient diet and then weaned to the same diet (group A in Table 1). Unlike cells obtained from rats whose intake of Cu was restricted during the lactation and postlactation periods (Fig. 3), the percentage of freshly isolated splenic mononuclear cells expressing IL-2R was not altered by postlactational Cu deficiency (Table 5). In vitro exposure of splenic mononuclear cells from both

DISCUSSION

One of the more interesting findings of this study was the presence of significantly fewer T (W3/13-J cells, but not B cells and macrophages, in the splenic mononuclear cell fraction isolated from Cu-deficient male rats. This reduction in the number of splenic T cells and the CD4+ (T helper) subclass was inde pendent of several complications often associated with severe Cu deficiency, e.g., anemia and altered mass of lymphoid tissues. In contrast, significantly decreased numbers of splenic CD8+ (T cytotoxic/ killer) cells were only observed in Cu-deficient male rats exhibiting lower hematocrit, reduced thymic mass and slight splenomegaly. The numbers of CD4 cells in peripheral blood of male rats (8, 9) and mouse splenocytes (5) are also decreased more readily than those of CDS cells by inadequate dietary Cu. It has recently been shown that when CDS cells are injected intrathymically, they proliferate prior to maturing into CD4 cells (13). Therefore, it has been suggested that the expression of CDS surface antigen may rep resent an intermediate event in the ontogeny of CD4 cells. The maturation and function of T cells is influ enced by thymulin and other hormones secreted by


FIGURE 3 Interleukin 2 receptor (IL-2R, OX39) and transferrin receptor (TfR, OX26) expression in freshly iso lated splenic mononuclear cells from control and Cu-deficient rats. Results (mean Â±SEM) represent the percentage of positively stained cells above background control. Male rats: +Cu (a - 13 solid bars); -Cu group A (n - 5 open bars); and, -Cu group B (n - 5 hatched bars); female rats: +Cu (n 12 solid bars) and -Cu (n - 6 open bars). Hematocrit and relative weight of lymphoid organs of Cu deficient males assigned to group A were similar to +Cu males, but these parameters were significantly different in group B Cu-deficient and +Cu rats (see Table 1). Significant differences (P < 0.05, Students-Newman-Keuls multiple range test) between treatment groups for the same sex are indicated by different letters above the error bars.

Cu-deficient and Cu-adequate male rats to ConA in creased the percentage of cells expressing IL-2R ap proximately 20-fold by 18 h. The relative densities of EL-2Ron splenic mononuclear cells from Cu-adequate and Cu-deficient rats were similar at 18 h (mean channel of log 8.0 Â±0.3 vs. 8.1 Â±0.5, respectively). After 66 h in culture, the percentage of mitogentreated splenic mononuclear cells from both groups of animals expressing IL-2R remained similarly elevated (Table 5), although receptor densities were higher than at 18 h (mean channel of log 11.7 Â±1.0 for +Cu cells vs. log 14.0 Â±3.3 for -Cu cells). Upon isolation, the percentage of splenic mononuclear cells from rats fed Cu-deficient diet for 5 wk after weaning that expressed TfR was about threefold that for cells from control rats (Table 5). Enhanced expression of TfR was maintained in cul tures of splenic MNC from Cu-deficient rats after 66 h incubation in mitogen-free medium. Exposure to ConA markedly increased TfR expression by cultured cells. The percentage of ConA-treated splenic mononuclear cells from Cu-deficient rats expressing surface TfR was higher than that for mitogen-treated cultures from Cu-adequate controls after 66 h; ex pression of TfR was similar in 18-h cultures of cells from Cu-deficient and Cu-adequate rats. Densities of TfR on ConA-treated splenic mononuclear cells from Cu-adequate and Cu-deficient rats were similar after 18 h (mean channels of log 6.7 Â±0.3 vs. log 7.7 Â±0.3, respectively) and 66 h (mean channels of log 10.2 Â± 1.2 vs. log 11.1 Â±0.9, respectively).

751

COPPER AND SPLENIC CELLULARTTY AND FUNCTION

TABLE 5 Concavavalin A-induced up-regalation of IL-2R and TfR an splenic mononuclear cells from control and copper-deficient male rats1 BL-2R

ConA

Incubation

+Cu

TfR

-Cu

cells2.3 __-++h018661866%

+Cu

-Cu

positive

O.r2.4 Â± 0.7'0.9 Â± 1.8'47.5 Â±

0.4'3.4 Â± 0.9-1.8 Â± 2.7*44.5 Â±

0.3'1.9 Â± 0.3'2.1 Â± 0.8'33.3 Â±

1.2*7.3 Â± 1.4"6.2 Â± 2.3V37.0 Â±

3.5'37.8 Â± Â±2.3'3.3

4.0*42.4 Â± Â±9.3Â«3.6

3.8'37.9 Â± Â±3.0-9.8

2.2'50.6 Â± Â±5.4k

thytnic epithelium (14, 15). Serum levels of thymulin are decreased in Cu-deficient rats (16). Perhaps, the apparently greater sensitivity of CD4 cells, when compared with CDS cells, to dietary Cu status indi cates that this subclass requires higher concentrations of Cu itself or thymulin than the CDS subclass for terminal differentiation. Other factors, including sex, also seem to modulate the level of dietary Cu re quired for T cell maturation, because the absolute and relative numbers of splenic mononuclear cells were similar in control and Cu-deficient female rats. Differ ential regulatory influences of sex steroids on immunocompetence are known (17, 18). The sex-dependent impact of Cu deprivation on splenic lymphoid cell subsets is comparable to the higher incidence of mor tality (19) and anemia (19, 20, this study) caused by Cu deficiency in male compared with female rats. The altered phenotypic profile of splenic mononuclear cells from Cu-deficient male rats is qualitatively similar to that of splenocytes from Cudeficient mice (5, 10). However, there are several noteworthy differences in the splenic cellularity of these two species in response to Cu deficiency. Lukasewycz et al. (5) found that the absolute number of splenic B cells was elevated twofold to threefold in Cu-deficient C58 mice. The numbers of T cells and the Lyt 1 and Lyt 2 subclasses were similar in spleens from control and Cu-deficient mice. Moreover, changes in the splenic B cell population due to Cu deficiency were observed in both sexes of mice. Rat splenic mononuclear cells and mouse splenocytes were isolated by density gradient centrifugation and differential centrifugation, respectively, prior to immunolabelling. Although the method of cell prep aration may have influenced the results, there may be other explanations for the differential effects of Cu deficiency on splenic lymphoid cells in rats and mice.

First, splenomegaly and anemia are generally conse quences of Cu deficiency in both sexes of mice (4, 5, 10, 21), and slight splenomegaly and anemia, if present, are only observed in Cu-deficient male rats (this study, 19, 20, 22). Second, histopathological ab normalities are localized in the medullary regions of the thymus of Cu-deficient rats and in the cortical region of the thymus of Cu-deficient mice (10, 22). The basis for these different consequences of Cu defi ciency in rats and mice is not clear. The attenuated response of splenic mononuclear cells from Cu-deficient male rats to nonspecific T and B cell mitogens agrees with previous reports for mice (4, 5) and rats (20, 23). As we recently observed with rat peripheral blood mononuclear cells (8, 9), the degree of suppression of mitogenic responsiveness by splenic mononuclear cells from Cu-deficient rats (i.e., 20-25% of control activity) markedly exceeds the decrease in the relative number of T cells (i.e., 80-85% control levels). Likewise, despite the presence of higher numbers of B cells per culture well, exposure of cells from Cu-deficient rats to LPS failed to elicit an enhanced or even normal response. PHAinduced reactivity of splenic mononuclear cells from female Cu-deficient rats was also significantly decreased, despite normal ratios of the various cell types. This lack of a simple relationship between changes in phenotypic profile and mitogenic reac tivity of splenic mononuclear cells is similar to results with splenocytes from Cu-deficient C58 mice (5). It has also been shown that splenocytes from Cudeficient mice are poor stimulators and responders in mixed lymphocyte reactions (24) and secrete decreased amounts of T cell growth factor in vitro (25). Taken together, these data suggest that Cu defi ciency inhibits either the ability of effector cells to properly transmit activation signals or target cells to


'Commercially weaned rats (five per group) were fed +Cu or -Cu diet for 5 wk before obtaining splenocytes. Splenic mononuclear cells (2 x 10*/mL) were cultured in RPMI 1640 containing 5% fetal calf serum either without (-) or with (+) ConA (5 Hg/mL) as indicated in text. Cells were harvested after 18 and 66 h of incubation, washed and stained with OX39 (IL-2R) and OX26 (TfR) monoclonal antibodies. Results represent mean Â±SEM. Significant differences (P < 0.05) between treatment groups at each time are indicated by different letters as superscripts, Student-Newman-Keuls multiple range test. Abbreviations used: ConA, Concanavalin A; IL-2R, interleukin-2 receptor; TfR, transfcrrin receptor.

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BALA ET AL.

including lymphocytes (12). The basis for and possible significance of altered expression of IL-2R and TfR by splenocytes from severely Cu-deficient rats is not known. In summary, the number and mitogenic reactivity of T cells isolated from the spleen of male rats were markedly decreased by dietary Cu deficiency. Whether this impaired DNA synthesis in the presence of mitogens only represents a defect in T cell function or abnormalities in the activities of APC and APC-T cell interactions is unknown. Our laboratory recently found that Cu deficiency attenuates the respiratory burst and candidacidal activity of peritoneal macro phages (3). Consequently, the effects of Cu deficiency on the ability of macrophages to process and present antigen to T cells are the focus of current studies.

ACKNOWLEDGMENTS We thank Beth Seidel and Richard Barr for tech nical assistance and Chris Woods for preparation of the manuscript.

LITERATURE CITED 1. Prohaska, J. R. &. Lukasewycz, O. A. (1989) Effects of copper deficiency on the immune system. In: Antioxidant Nutrients and Immune Functions. (Benedict, A., Phillips, M. & Tengerdy, R., eds.) pp. 123-143, Plenum Press, New York, NY. 2. Jones, D. G. &. Suttle, N. R. (1981) Some effects of copper deficiency on leukocyte function in sheep and cattle. Res. Vet. Sci. 31: 151-156. 3. Babu, U. &. Failla, M. L. (1990) Respiratory burst and candida cidal activity of peritoneal macrophages are impaired in copper deficient rats. J. Nutr. 120: 1692-1699. 4. Prohaska, J. R. &. Lukasewycz, O. A. (1981) Copper deficiency suppresses the immuni- response in mice. Science 213: 559-561. 5. Lukasewycz, O. A., Prohaska, J. R., Meyer, S. G., Schmidtke, J. R., Hatfield, S. M. &. Marder, P. (1985) Alterations in lym phocyte subpopulations in copper-deficient mice. Infect. Immun. 48: 644-647. 6. Mulhern, S. A., Raveche, E. S., Smith, H. R. &. Lai, R. B. (1987) Dietary copper deficiency and autoimmunity in the NZB mouse. Am. J. Clin. Nutr. 46: 1035-1039. 7. Failla, M. L., Babu, U. S. Seidel, K. E. (1988) Use of immunoresponsiveness to demonstrate that the dietary re quirement for copper in young rats is greater with dietary fructose than dietary starch. J. Nutr. 118: 487-496. 8. Bala, S., Failla, M. L. &. Lunney, J. (1990) Phenotypic and functional alterations in peripheral blood mononuclear cells of copper deficient rats. Ann. N.Y. Acad. Sci. 587: 283-285. 9. Bala, S., Failla, M. L. &. Lunney, J. (1990) T-cell numbers and mitogenic responsiveness of peripheral blood mononuclear cells are decreased in copper deficient rats. Nutr. Res. 10: 749-760. 10. Mulhern, S. A. A. Koller, L. D. (1988) Severe and marginal copper deficiency results in a graded reduction in immune, status in mice. J. Nutr. 118: 1041-1047. 11. American Institute of Nutrition. (1977) Report of the AIN Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 107: 1340-1348.


receive and respond to these signals, or some combi nation of impaired activities of both effector and target cells. Assessment of mitogenic responsiveness monitors a late event in T cell activation, e.g., DNA synthesis. Cellular events that precede the clonal expansion of T lymphocytes include antigen processing and the pre sentation of antigenic fragment in association with Class n major histocompatibility complex (MHC) molecules on the surface of APC (26), cytokine se cretion by APC and target T cells (27), and the ex pression of IL-2R and TfR on the surface of T cells (12, 28). To initiate studies aimed at elucidating the basis for the impaired mitogenic reactivity of splenic mononuclear cells from Cu-deficient rats (postlactational model of Cu deficiency), we examined the in vitro expression of IL-2R and TfR. The results show that the kinetics and degree of up-regulation of these receptors after exposure to nonspecific mitogen were not altered by Cu deficiency. This apparent un coupling of receptor expression and DNA synthesis resembles a recent report that the mitogenic reac tivity of splenocytes from aged rats is impaired despite normal expression of IL-2R and secretion of IL-2 (29). The authors suggested that the affinity of EL2R for IL-2 may be altered. Cu deficiency has also been reported to decrease the amount of IL-2 released by mouse splenocytes in vitro (25). This observation and our data on IL-2R expression suggest that Cudeficient T cells may secrete sufficient levels of DL-2 required for expression of the IL-2R, but these levels may be insufficient to fully activate later events re quired for normal induction of DNA synthesis. The percentage of freshly isolated splenic mononuclear cells expressing IL-2R and TfR was in creased in male rats subjected to restricted intake of Cu during the lactation and postlactation periods. Increased numbers of spontaneously cycling cells [S+G2phase (10)] and blastoid-like and, possibly, im mature cells (5) have been independently reported among splenocytes from Cu-deficient mice. Activated and immature lymphocytes express higher levels of IL-2R and TfR than quiescent mature cells (12, 28, 30). Also, activation of a limited percentage of total population might be expected if experimental animals had a subclinical infection; susceptibility to infection is increased by Cu deficiency (1). Because receptor levels were not altered in splenic mononuclear cells from Cu-deficient female rats, it is also possible that the altered splenic cellularity in Cu-deficient male rats accounts for the observed change in the per centage of cells expressing the IL-2R and TfR. The increased expression of TfR by splenocytes from Cudeficient male rats may also be secondary to altered iron status, because the degree of expression of TfR and hematocrit were inversely related. Decreased intracellular iron is associated with enhanced ex pression of TfR on the surface of many cell types,

COPPER AND SPLENIC CELLULARTTY AND FUNCTION

20. Kramer, T. R., Johnson, W. T. &. Briske-Anderson, M. (1988) Influence of iron and the sex of rats on hematological, bio chemical and immunological changes during copper defi ciency. J. Nutr. 118: 214-221. 21. Lukasewycz, O. A &. Prohaska, J. R. (1981) Immunization against transplantable leukemia impaired in copper-deficient

mice. J. Nati. Cancer Inst. 69: 489-493. 22. Koller, L. D., Mulhem, S. A, Frankel, N. C., Steven, M. G. &. Williams, J. R. (1987) Immune dysfunction in rats fed a diet deficient in copper. Am. J. Clin. Nutr. 45: 997-1006. 23. Davis, M. A, Johnson, W. T., Briske-Anderson, M. &.Kramer, T. R. (1987)Lymphoid cell functions during copper deficiency. Nutr. Res. 7: 211-222. 24. Lukasewycz, O. A, Kolquist, K. L. & Prohaska, J. R. (1987) Splenocytes from copper-deficient mice are low respondcrs and weak stimulators in mixed lymphocyte reactions. Nutr. Res. 7: 43-52. 25. Lukasewycz, O. A. &.Prohaska, J. R. (1990) Immune response in copper deficiency. Ann. N.Y. Acad. Sci. 587: 147-159. 26. Unanue, E. R. Â¿X Cerottini, J. C. (1989) Antigen presentation. FASEBJ. 3: 2496-2502. 27. Balkwill, F. R. &. Burke, F. (1989) The cytokine network. Immunol. Today 10: 299-303. 28. Waldman, T. A (1986) The structure, function and expression of interleukin-2 receptors on normal and malignan* lympho cytes. Science 232: 727-732. 29. Holbrook, N. J., Chopra, R. K., McCoy, M. T., Nagel, J. E., Powers, D. C., Adler, W. H. & Schneider, E. L. (1989) Ex pression of interleukin-2 and the interleukin-2 receptor in aging rats. Cell. Immunol. 120: 1-9. 30. Tentori, L., Longo, D. L., Zuniga-Pflucker, J. C., Wing, C. & Kruisbeek, A M (1988) Essential role of the interleukin-2 receptor pathway in thymocyte maturation in vivo. J. Exp. Med. 168: 1741-1747.


12. Pelosi-Testa, E., Samoggia, P., Giannelk, G., Montesoro, E., Caravita, T., Salvo, G., Camagua, A., Isacchi, G., Testa, U. &. Peschle, C. (1988) Mechanisms underlying T-lymphocyte acti vation: mitogcn initiates and IL-2 amplifies the expression of transferrin receptors via intracellular iron pool. Immunology 64: 273-279. 13. Nikolic-Zugic, J. &.Sevan, M. J. (1988)Thymocytes expressing CDS differentiate into CD4* cells following intrathymic injec tion. Proc. Nati. Acad. Sci. USA 85: 8633-8637. 14. Paure, G., Bene, M. C., Tamisier, J. N., Gaucher, A. & Dukeille, J. (1984) Thymulin (FTS-Zn) induced in vitro modu lation of T-cell subset markers on lymphocytes from arthritis and systemic lupus erythematosus patients. Int. J. Immunopharmacol. 6: 308-314. 15. Savino, W. & Dardenne, M (1984) Thymic hormone con taining cells. VI. Immunohistologic evidence for the simul taneous presence of thymulin, thymopoietin and thymosin alpha 1 in normal and pathological human thymuses. Eur. J. Immunol. 14: 987-991. 16. Vyas, D. & Chandra, R. K. (1983) Thymic factor activity, lymphocyte stimulation response and antibody producing cells in copper deficiency. Nutr. Res. 3: 343-349. 17. Grossman, C. J. (1985) Interactions between the gonadal steroids and the immune system. Science 227: 257-261. 18. Sthoeger, Z. M., Chiorazzi, N. &. Lahita, R. G. (1988) Regu lation of the immune response by sex hormones. I. In vitro effects of estradial and testosterone on pokeweed mitogeninduced human B-cell differentiation. J. Immunol. 141: 91-98. 19. Fields, M., Lewis, C., Scholfield, D. J., Powell, A. S., Rose, A. J., Reiser, S. fit Smith, J. C. (1986) Female rats are protected against the fructose induced mortality of copper deficiency. Proc. Soc. Exp. Biol. Med. 183: 145-149.

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