Intracellular zinc in chronic lymphocytic leukemia

CLINICAL

IMMUNOLOGY

Intracellular

AND

24, 26-32 (1982)

IMMUNOPATHOLOGY

Zinc in Chronic

Lymphocytic

R. J. KANTER,~ K. R. RAI, F. MUNIZ, B. MICHAEL, A. SAWITSKY~

Leukemia’ J. BALKON,

AND

In this study, we measured the intracellular zinc (Zn) concentration of normal blood lymphocytes and of B-cell chronic lymphocytic leukemia (CLL) lymphocytes using anodic stripping voltometry analysis of Ficoll-Hypaque-isolated cells. A significant difference was found between the mean Zn concentration of normal lymphocytes (IO. 1 _f 0.8 ng/106 cells) and those from patients with CLL (4.3 ? 0.4 ng) (P < 0.01). Patients with late-stage CLL had significantly less intracellular lymphocyte Zn (ng/106 cells) than did early stage patients, 3.5 _t 0.4 and 5.8 -t 0.8, respectively (P < 0.05). Overall, in the total patient populations, there was a significant inverse correlation between the absolute peripheral blood lymphocytosis and Zn concentration/106 cells. Concentration of Zn in serum was within the normal range among all patients. There was no correlation between lymphocyte Zn levels and proportion of B- and T-lymphocyte subpopulations. Exogenous Zn did not amplify mitogen response of lymphocytes. The implications of these findings in lymphocyte function in CLL are discussed.

INTRODUCTION

Zinc has been implicated as an essential nutrient in the regulation of the immune response (1). Severe Zn deficiency in animals and in man has been associated with defects in cellular immunity. Dietary zinc deficiency may impair helper T-cell function in the adult mouse (2). Increased dietary zinc enhances the lymphocyte phytohemagglutinin (PHA) stimulation index. III vitro zinc ions are mitogenic to normal lymphocytes in the human (3). The present study was undertaken to determine the intracellular zinc concentration in normal lymphocytes and in lymphocytes isolated from patients with B-cell chronic lymphocytic leukemia (CLL). These findings have been correlated with clinical stage (4) and progression of disease. MATERIALS

AND METHODS

Heparinized venous blood (10 units heparimml) was collected aseptically from 78 patients with stages O-IV CLL and from 20 normal control subjects. Complete blood counts were determined and the following detailed lymphocyte studies were performed. ’ Supported by grants from the National Leukemia Association, Inc., the United Leukemia Fund. the Helena Rubinstein Foundation, the Rosenstiel Foundation. the Wayne Goldsmith Leukemia Foundation, and Research Grant CA-I 1028 from the National Cancer Institute. * Present address: Technicon Corp., Tarrytown, N.Y. 3 To whom reprint requests should be addressed at the Long Island Jewish-Hillside Medical Center. 26 0090.1229/82/070026-07$01.00/O Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

ZINC

IN

CHRONIC

LYMPHOCYTIC

LEUKEMIA

27

1. Lymphocyte separation from peripheral blood samples. Peripheral blood lymphocytes were isolated by the density gradient technique of Boyum (5). The heparinized blood was diluted with an equal volume of calcium and magnesium free Hank’s balanced salt solution (HBSS) (GIBCO). The diluted blood was layered over Ficoll-Hypaque and centrifuged at 4008 for 20 min at room temperature. The resultant layer of mononuclear cells was aspirated from the gradient interface and washed three times with HBSS. The cells were resuspended to a concentration of 2 x lo6 mononuclear cells/ml. Cell viability was assessed by trypan blue dye exclusion technique and was routinely greater than 95%. This mononuclear cell suspension was used for all lymphocyte zinc analyses, surface marker assays, and stimulation studies. 2. Zinc analysis. Aliquots of the mononuclear cell preparations (normal or CLL) were treated with 0.83% ammonium chloride to lyse residual erythrocytes and were then washed three times with phosphate-buffered saline (pH 7.2). After the final wash, counted lymphocyte suspensions were pelleted in acid-washed digestion tubes and stored frozen at -20°C. The samples were digested to dryness with 0.3 ml of certified trace metal-free perchloric acid (G. Frederick Smith) at 210°C. The digestion residue was reconstituted in trace metal-free acetate buffer (pH 6.0, I M sodium acetate, 0.2 M sodium chloride). Total zinc content per tube was determined on a Model 2011 anodic stripping voltmeter (Environmental Science Associates). Zinc was electrochemically plated from the acetate buffer onto a heavy mercury-coated graphite electrode for 5 min in a nitrogen atmosphere. Plating potential was - 1380 mV. After plating, the zinc was stripped from the mercury coat anodically at 60 mV/sec. Stripping was terminated at -800 mV. The electrode was washed copiously with distilled deionized water prior to addition of the next sample. Voltamograms were recorded on a Perkin-Elmer 56 recorder and measurement of zinc peaks performed by a peak height method. Calibration for total zinc content per digestion tube was established by assaying known quantities of zinc in the range of 50-500 ng per tube with each run of lymphocyte preparations. Lymphocyte zinc concentration was determined by dividing total zinc per tube by the number of cells digested. This result was expressed as nanograms zinc per lo6 lymphocytes (ng Zn/106 lymphocytes). Serum zinc concentration of both CLL patients and normal control subjects were determined using atomic absorption spectrometry . Seven CLL patients were given pharmacologic doses of oral zinc sulfate (220 mg four times a day). The lymphocyte Zn content of blood lymphocytes from these patients was assayed after varying periods (4-26 weeks) on Zn therapy. 3. Lymphocyte subpopulations. B- and T-lymphocyte populations were determined by the following techniques: (a) T cells: Aliquots of the mononuclear cell preparations (normal or CLL) were used to determine E-rosette-forming cells using a method previously described (6). Cells with three or more adherent sheep red cells were scored as positive. At least 200 cells were counted and the results expressed as the percentage of rosetting cells of the total cells counted. Rosette-forming cells were also determined in

28

KANTER

ET AL.

the presence of varying micromolar concentrations of zinc chloride. Helper and suppressor T-cell populations were not assayed separately. (b) B cells: Surface immunoglobulin-bearing cells (slg) were determined as previously described (6). Aliquots of lymphocyte suspensions were stained with the (Fab), fragment of polyvalent fluorescein isothiocyanate-conjugated antiimmunoglobulin serum (Kallstad Laboratories). A minimum of 200 cells were counted using a Leitz Orthoplan microscope equipped for epifluorescence illumination. Results were expressed as a percentage of tagged cells of the total cells counted. Lymphocytes bearing a membrane receptor for C, were assayed according to the erythrocyte-antibody-complement (EAC) method of Bianco et al. (7) as previously described (6). Lymphocytes with three or more adherent EAC indicator cells were scored as positive. Results were expressed as a percentage of positive cells of the total cells counted. 4. Lymphocyte blastogenic studies. Aliquots of the total lymphocyte population were studied for their ability to respond to stimulation with phytohemagglutinin (PHA) (Burroughs Wellcome Co.), or pokeweed mitogen (PWM) (GIBCO). Concurrent controls were studied without any mitogen. Zinc chloride (0.2 mM) was added to both mitogen-stimulated and control cultures to assess the effect of exogenous zinc on lymphocyte blastogenic response. These studies were done using a modification of the method of Strong et al. (8). 5. Statistical analysis. The differences between mean zinc concentrations were tested by Student’s t-test for independent samples. Correlation coefficients (Pearson’s Y) were calculated from a plot of the logarithmic transformation of absolute lymphocyte count in blood versus Zn concentration/106 cells. RESULTS

I. Lymphocyte

Zinc Concentration

Lymphocyte zinc concentrations of 78 CLL patients and 20 control subjects expressed as nanograms/lo6 lymphocytes are summarized in Table 1. A significant difference was found between the mean Zn concentration in lymphocytes from normal individuals (10.1 -+ 0.8 ng) and those from patients with CLL (4.3 + 0.4 ng) (ZJ < 0.01). When the zinc concentration/lO’j lymphocytes was studied by clinical stage of disease, patients with late stage disease had significantly less lymphocyte Zn (P < 0.05) than did early stage patients as detailed in Table 2. TABLE LYMPHOCYTE

ZINC LEUKEMIA

Subject

N

CLL

78

Control

20

CONCENTRATION PATIENTS

AND

1 IN CHRONIC CONTROL

LYMPHOCYTIC

SUBJECTS

Lymphocyte Zn (ngi106 cells)

P

4.3 -t 0.40 co.01

” Mean ? SEM.

10.1 t 0.8

ZINC IN CHRONIC

LYMPHOCYTIC TABLE

LYMPHOCYTE

ZINC

STAGE

2

CONCENTRATION

CHRONIC

29

LEUKEMIA

IN EARLY-

LYMPHOCYTIC

AND

LATE-

LEUKEMIA

Subjects

N

Lymphocyte Zn (ng/106 cells)

Early stages (0. 1)

43

5.8 2 0.8"

P

-

co.05 Late stages (II. III. IV)

35

3.5 5 0.4

’ Mean + SEM.

When the lymphocyte Zn concentration of the 78 CLL patients was analyzed according to the absolute peripheral blood lymphocyte count, the Zn concentration/106 lymphocytes was found to be inversely related to the absolute lymphocyte count as shown in Fig. 1. Logarithmic (In) transformation of this data demonstrates a linear relationship between these variables with a progressive increase in the slope of the curve as disease stage advances. There is a significant correlation between absolute lymphocytosis and Zn concentrationDO lympho-

170% x 2 5

WIlSO140-

g

ml-

: A

120 -

g 0 &Y 2 5 I I z i 2

llOloo90ED70 EO50403020 -

1

2

3

4

NANOGRAMS

5

6

ZINC/IO’

7

8

9

10

11

1 12

LYMPHOCYTES

FIG. I. The concentration of zinc in nanograms per lo6 lymphocytes is plotted against the absolute peripheral blood lymphocyte concentration expressed as cells per microliter. As the peripheral blood lymphocyte concentration falls, there is an increase in intracellular zinc concentration.

30

KANTER

ET

AL.

cytes in the total patient population and in each stage of disease except stage II (Table 3). No change in lymphocyte Zn concentration was observed in the seven patients who received supplementary oral zinc sulfate. 2. Serum Zinc Concentration When serum zinc concentrations were determined, there were no significant differences found between the serum Zn concentration of CLL patients and those of normal subjects. All the serum Zn values fell within the normal range of 70- 130 /-ddl. 3. Lymphocyte

Subpopulations

No correlation was found between Zn concentration and either T- or Blymphocyte subpopulations. Rosette-forming assays performed in the presence of varying concentrations of exogenous Zn did not significantly increase rosette information in either CLL or normal lymphocytes. 4. Lymphocyte

Blastogenic

Studies

The addition of 0.2 m&f exogenous Zn had no effect on the response pattern obtained from CLL lymphocytes on any day during the lo-day culture period in either stimulated or unstimulated cultures. However, normal lymphocytes not stimulated with either PHA or PWM, showed a blastogenic response to exogenous Zn at 6-8 days of culture. Exogenous Zn did not amplify the PHA or PWM response of these normal lymphocytes. DISCUSSION

The ubiquitous distribution of Zn, its role in enzyme function, and in subcellular structures has prompted numerous studies of the relationship of Zn to the immune response (I-3). The well-documented in vivo and in vitro immune defects seen in patients with CLL suggested that the CLL lymphocyte might provide additional information on the role of Zn ions in immune function (9). In the present study, no correlation was found between lymphocyte Zn content and lymphocyte T- and B-cell subpopulations in either CLL patients or in normal subjects. Although McMahon et al. (10) have reported that incubation with exogenous ZnCL, enhances SRBC rosette formation by lymphocytes from either normal subject or CLL patients, we did not observe such an effect. TABLE CORRELATION ZINC

Stage 0

I II III/IV All stages

COEFFICIENTS

BETWEEN

CONCENTFWTION/~~~

N

18 25 20 15 78

CELLS

3

ABSOLUTE

LYMPHOCYTE

ACCORDING

TO CLINICAL

Correlation

coefficient

-0.4435 -0.4884 -0.4398 -0.4069 -0.7607

COUNT STAGE

IN BLOOD

AND

OF CLL

P <0.05 <0.05 NS
ZINC

IN

CHRONIC

LYMPHOCYTIC

LEUKEMIA

31

Dennes et al. (11) in 1961 reported that Zn concentration in leukocytes from patients with CLL was decreased as compared to normal leukocytes. Our finding of decreased lymphocte Zn concentration in CLL patients correlates with the earlier observations of Dennes et al. (11) on unseparated leukocytes which included polymorphonuclear cells. Lymphocytes from the patients with CLL have an impaired and delayed blastogenic response to mitogen stimulation. Since Zn is an essential factor in DNA and protein synthetic pathways, it is possible that decreased Zn content of lymphocytes in CLL acts as a contributing factor in their poor blastogenic response. However, we failed to demonstrate resotration of lymphocyte blastogenic response by adding exogenous Zn in the cultures. A likely explanation for this observation is the defective in vitro uptake of Zn by CLL lymphocytes (12). Gunther et al. (13) demonstrated that Zn content of leukemic lymphocytes was reduced in both the nucleus and the cytoplasm, and that PHA stimulation had no effect on Zn binding proteins within the cell. The decline in mean lymphocyte Zn concentration with disease progression in CLL is as yet unexplained, and may only reflect the strong negative correlation of lymphocyte Zn concentrations to the lymphocytosis associated with increasing clinical disease. Similar inverse correlation between leukocyte Zn content and number of leukocytes in blood in CLL (but not in chronic myelocytic leukemia) was earlier noted by Dennes et al. who could offer no explanation for this finding. To us, this finding suggests the possibility that there is a limited pool of Zn available for incorporation into cells. Pharmacologically active doses of zinc sulfate did not increase or alter the lymphocyte Zn concentration in seven CLL patients. This observation is not unexpected in the light of the normal serum Zn values obtained in these patients and the report of Fuchwans et al. (12) of defective uptake of radiolabeled Zn by CLL lymphocytes. It appears that lymphocytes in CLL differ from lymphocytes in other malignancies because in contrast to CLL where low intracellular Zn was noted in all patients, less than half of patients with non-Hodgkin’s lymphoma and nonhematological malignancies showed low intracellular zinc values, and only an occasional patient with acute leukemia or myeloproliferative syndrome had a low value (14). We have found a decrease in the intracellular lymphocyte Zn concentration in B-cell CLL lymphocytes which is not associated with a serum Zn deficiency and which is not corrected or altered by oral Zn supplementation. Changes in the Tand B-cell ratio did not affect the intracellular Zn concentration values. However, an increasing absolute peripheral blood lymphocytosis was associated with a decrease in intracellular Zn content. Further studies of isolated T and B lymphocytes and of T-cell subpopulations will be required to define the effect of cellular maturity and the differentiation status of the leukemic T and B cell in CLL to the functional role of Zn in these lymphocytes. REFERENCES I. Good. 2. Fraker.

R. A. and Fernandes, P. J.. and Luecke.

G., Clin. Bull. 9, 63, 1979. R. W., Fed. Proc. 36, 1176. 1977.

32

KANTER

ET AL.

3. Kirchner, H., and Rtihl, H., Exp. Cell Re.7. 61, 229, 1970. 4. Rai, K. R., Sawitsky, A., Cronkite, E., Chanana, A., Levy, R., and Pasternack, B.. Bjmd 46, 219, 1975. 5. Boyum, A., Scand. J. C/in. Lab. Invest. 21, 77, 1968. 6. Kanter, R. J., Freiberger, I. A.. Rai. K. R.. and Sawitsky, A., C/in. Inrtn~rtto/. Itrlmrtnopat/l(t/. 12, 351, 1979. 7. Bianco. C., Patrick, R., and Nussenzweig, V., J. Exp. Med. 132, 702, 1970. 8. Strong, D. M., Ahmed, A. A., Thurman. G. B.. and Sell, K. W., J. Immune/. Mrrhods 2, 279, 1973. 9. Rai, K. R., and Sawitsky, A., Contemp. Hematol./Ckol. 2, 227, 1981. 10. McMahon, L. J., Montgomery. P. W., Grodowski. A., Woods, A., and Zuboski, G., Immu&. Commtrn. 5, 53. 1976. 11. Dennes, E., Tupper, R., and Wormall, A., Bkxhetn. J. 78, 578, 1961. 12. Fuchswans, W., Binder, B., Hocher. P., and Binder, C., Wein. Klirz. Wijchettsc/tr. 11, 389, 1977. 13. Gunther, T., Averdunk, and Rtihl, H., 2. Immunol. For.~cl~. 155, 269, 1979. 14. Kanter, R. J., Rai, K. R.. Balkon, J., Muniz, F.. Michael, B., and Sawitsky, A., Blood 54 (Suppl. 1). IOla, 1979. Received September 16. 1981; accepted with revisions December 16. 1981