Tissue zinc dynamics during the immune reaction in mice

@Copyright 1998 by Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/98/6502-0097 $11.00

Tissue Zinc Dynamics During the Immune Reaction in Mice

DONATELLA VERBANAC,1 CEDOMILA MILIN, BISERKA RADO~EVI~-STA~IC,*'2 ZLATKO TROBONJA~A, 2

ROBERT DOMITROVI(~,~ JASM NKA GIACOMETTI, M~RIJA PETKOVIC, 2 MiRA ( uK, 2 ZLATKO CIGANJ, 3

JASMINKA RUP(~I(~, ~ AND DANIEL RUKAVlNA 2

IDepartment of Chemistry and Biochemistry, and 2Department of Physiology and Immunology, Medical Faculty of Rijeka,

B. Branchetta 22, 51 000 RIJEKA, Croatia; and 3lNA-Rafinery Urinj, 51 000 Rijeka, Croatia

Received February 18, 1998; Accepted April 15, 1998

ABSTRACT

Owing to the importance of zinc for the functioning of the immune system, the role of endogenous Zn, located both in lymphoid and nonlymphoid organs, was investigated during the standard humoral and cellular types of immune response. For this purpose, the dynamics of hepatic, thymic, splenic, and renal Zn con- tent was determined in mice sensitized with (a) sheep red blood cells and (b) semiallogeneic lymphocytes during the local host vs graft reaction (HVGR). The data obtained by ion-coupled plasma spectrometry revealed that the humoral type of immunity is characterized by a significant increase of Zn concentration in the liver and in the thymus. Simultaneously, linear regression analysis showed that the generation of plaque-forming cells in the individual mouse was highly positively correlated with Zn concentration in the liver (r = 0.897), and spleen (r = 0.833), and negatively with Zn concentration in the thymus (r = -0.624). Similar relationships between the intensity of local immune reaction and tissue Zn levels were found in local HVGR at the fifth day in the liver and spleen (r = 0.861 and r = 0.695, respectively), at the seventh day in the thymus (r = -0.797), and at the tenth day in the liver (r = -0.859).

*Author to whom all correspondence and reprint requests should be addressed.

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The data emphasize the necessity of Zn for the development of normal immune response and point to the existence of a Zn- dependent hepato-thymic axis during the humoral and cellular types of immune reactivity.

Index Entries: Zinc; ICP spectroscopy; atomic absorption; zinc in tissue; humoral and cellular types of immunity; plaque-forming cells generation; local host vs graft reaction.

INTRODUCTION

The special properties of the zinc cation and its unique chemistry and availability permit basic structural, catalytic, and regulatory functions of Zn in a number of biological systems (1). In the early 1960s, zinc was first identified as essential and, then, as an integral part of more than 300 enzyme systems that are involved in major metabolic pathways (2). Its importance in growth and development processes, reproduction, respira- tion, DNA synthesis and neurosensory and hormonal functions is now widely accepted (3). Additionally, the extremely important interrelation- ship between zinc and the immune system has been reported (for review, see refs. 4 and 5). Zinc is necessary for the development and maintenance of lymphatic functions. Its deficiency is followed by thymic atrophy, lym- phopenia, and alterations in the proportions and functional activities of the various subsets of lymphocytes and mononuclear phagocytes. As a result, in experimental models the changes of antibody and cell-mediated immunity might be induced, which often correlate with similar findings in human states of zinc deficiency. On the other hand, a number of evidence shows that supplementation with Zn, both in vivo and in vitro, may correct the majority of observed changes in immune function (4,5).

Owing to the obvious link between Zn and immunity, in this study an attempt was made to explore the behavior of endogenous Zn, located both in lymphoid and nonlymphoid organs, during the standard types of immune response. For this purpose, hepatic, thymic, splenic, and renal zinc concentrations were determined by a recently published method (6) for tissue disintegration and inductivity coupled plasma spectrometry (ICPS) for trace metal measurement in mice: (1) sensitized with sheep red blood cells (SRBC) and (2) subjected to local host vs graft reaction (HVGR). In each mouse, the correlation between the tissue Zn concentration and the intensity of immune reaction was also made.

MATERIAL AND METHODS

Mice

Male BALB/c (H-2d) and F1 (BALB/c x CBA (H-2k) mice, aged 2-3 mo were obtained from our own breeding colony. They were kept

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Tissue Zinc Concentration and Immune Response 99

in standard laboratory conditions, on water ad libitum, and a natural day-night cycle.

Plaque-Forming Cells Response

Mice were immunized by intraperitoneal injection of SRBC and 4 d later hemolytic plaque-forming cells (PFC) were determined in the spleen by the method of Cunningham and Szenberg (7).

Host vs Graft Reaction

The HVGR was measured by the popliteal lymph node (PLN) assay (8). For this purpose, semiallogeneic splenocytes (5 x 106), obtained from F1 (BALB/c x CBA) mice were injected into the right footpad of BALB/c mice. After 5, 7, and 10 d, the draining PLNs were isolated and weighed. The difference in weight was determined from the right and left PLN. Systemic effects of local HVGR were evaluated by determination of cellularity and phenotypic profile of cells in the spleen.

Cell Separation

Murine spleens or popliteal lymph nodes were aseptically removed from donor mice and gently pressed through fine stainless- steel screens in RPMI 1640. Red blood cells were lysed by Tris- buffered ammonium chloride for 5 min. After washing, the suspension of cells was filtered through fine nylon mesh, resuspended in complete media, and adjusted to the desiderated final concentration for PFC response, for sensitization of parental strain of mice, or for cytofluoro- metric (FACS) analysis.

Cyto f luoromet r i c A n a l y s i s

The number of CD4 § CD5 § or CD8 § cells in a suspension of freshly prepared spleen cells was evaluated on a fluorescence-acti- vated cell sorter (FACScan, Becton Dickinson, Immunocytometry Sys- tems, Mountain View, CA) using FACScan Research Software. Propidium iodide (1 ~tg/mL)-stained dead cells were excluded by electronic gat- ing. By the use of phycoerytrin or fluorescein isothiocyanate-conju- gated monoclonal antibodies (Becton Dickinson), 2 x 104 cells were analyzed. As controls, mouse IgG2b-PE, mouse IgG2a-PE, mouse IgG1- FITC, and mouse IgG2a-FITC antibodies were used (Coulter Clone, Coulter Immunology, Hialeah, FL).

Tissue Sample Preparations for Mineralization

From the mice sacrificed by exsaguination, the liver, thymus, spleen and kidney were carefully removed using plastic instrumentation on the


100 Verbanac et al.

fourth day after challenge with SRBC (when an antibody-dependent type of immunity was tested) or at the fifth, seventh, and tenth day after sensitization of mice with semiallogeneic lymphocytes (when the cell-mediated type of immunity was tested). To avoid possible diurnal variability, all operations were done between 8:00 and 9:00 AM.

The samples of tissues were selected and prepared as previously described (6). Briefly, 100-450 mg of liver, spleen, and kidney or 20-50 mg of the thymus were dried at 105~ for 5 h. After that, 3 mL of conc. HNO3 ("Kemika" d. d., Zagreb, Croatia) and 0.5 mL of 30% H202 ("Kemika" d. d.) were added. The microwave digestion occurred in the MLS-1200 Mega, Microwave Digestion System with MDR Technology, under the following conditions: 5 min at 300 W, 30 s at 0 W, 5 min at 600 W, and 1 min of ventilation. After cooling the samples for 15 min in cool water, the samples were transferred into the 10-mL flasks and the destillated/demineralizated water was added to the mark.

Inductively Coupled Plasma Spectrometry (ICP)

Liquid samples were introduced into the apparatus by pneumatic nebulization, which is the most popular method because of its desirable characteristics (relatively good stability, rapid changeover, and no mem- ory effects with specificity in operation). Measurements were performed using a Philips PU 7000-ICP Spectrometer (Scientific Analytical Division, Cambridge, UK), using the method outlined in ASTM D-19756-91 (power 1 kW, coolant 12 L/min, nebulizer 38 psi) and the wavelength of 213.856 nm. A single zinc standard (Merck, Darmstat) of 1000 ppm was diluted to obtained appropriate standard concentrations. The lower detection limit was 0.05 ppm and relative standard deviation was 3%.

Statistical Analysis The data were analyzed using the Sigma Plot Scientific Graphing

System, Version 4.03. The statistical significance was calculated by the two-tailed Student's t-test for unpaired samples, by a test of correlation, and by linear regression analyses using statistical software. Linear regression equations were generated using a least-square method and analyzed for differences of covariance. A p < 0.05 was considered significant. Data are reported as mean _+ SEM, unless otherwise noted.

RESULTS

Humoral Type of Immunity

The determination of tissue zinc concentrations in the thymus, spleen, liver, and kidney in mice sensitized with fiRBC and in the control



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Fig. 1. Tissue zinc changes induced by ip sensitization of mice with SRBC. Control group of mice was injected with saline solution. Each group consisted of four to six mice. Values are mean _+ SEM.

group of mice (injected with the same amount of saline) revealed that during the time of maximal plaque-forming cells generation, the concentration of endogenous zinc in thymus and liver increased significantly (Fig. 1). Furthermore, by linear regression analysis, a high positive correlation in the spleen (r = 0.833) and liver (r = 0.897) and a high negative correlation in the thymus (r = -0.624) between the PFC generation, the zinc concentration in particular organ was found (Fig. 2). Simultaneously, no correlation between the zinc value in the kidney, which served as the control organ, and PFC production in the spleen was observed (data are not shown).

Cellular Type of Immunity

The local HVGR, induced by the sensitization of mice by semiallogeneic cells (Fig. 3), was characterized by the slight increase in the weight of regional popliteal lymph nodes and by significant changes in the pro- portion of T- and B-lymphocyte subpopulations in the spleen (increase of


102 Verbanac et al.

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d i f f e r e n t o r g a n s of m i c e s e n s i t i z e d w i t h SRBC.



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CD4 § and CD8 § in spleen and decrease of Ig § cells). Simultaneously, an early (at the fifth day) slight decrease in the concentration of zinc in the spleen (p = 0.05) and a marked late (at the tenth day) fall in concentration of Zn in the liver (p < 0.001) were noted (Fig. 4). Moreover, when in the individual mouse, the intensity of local increase in the PLN weight was correlated with zinc concentration in the liver and lymphatic organs (Fig. 5), highly significant correlations were found on the fifth day of local HVGR in the spleen (r = +0.695) and in the liver (r = +0.861), on the


104 Verbanac et al.

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seventh day in the thymus (r = -0.797), and on the tenth day in the liver (r = -0.859).

D I S C U S S I O N

As expected, the data have shown that both humoral and cellular types of immune reaction may induce significant changes in tissue Zn dynamics. The greatest changes were observed in lymphatic organs, but also in the liver, which obviously must be considered as a site of activation of mononuclear lymphatic cells and extrathymic hematopoe-



Fifth day Seventh day SF'LEEN y = -9.093 + A5286 * x T I tYMUS y = 123.86 - 1.672 * x

Correlation: r = .69594 Correlation: r - -.7979 55 ..... 90 -

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Tenth day E ,-- LIVER y = -64.53 + 1.7157 * x LIVER Y = 1293.0 - 42.33 * x

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Zinc concentration (I.tg/g wet weight)

Fig. 5. Correlation (r) between intensity of local immune reaction (expressed as net increase in the weight of popliteal lymph node [PLN]) and Zn concentration in different organs during the HVGR.

sis, and as an important regulator of the Zn turnover, owing to its central role in the metallothionein (MT) metabolism in the body. The data obtained by linear regression analyses show that early phases of both types of immune reaction (PFC generation, estimated 4 d after sensitization of mice with SRBC and the net increase of popliteal lymph nodes after inoculation of semiallogeneic lymphocytes) are highly positively correlated with zinc concentration in the liver and in the spleen. The data therefore suggest the role of zinc ions and/or chelated zinc, particularly in these organs, for the normal process of T- and B-cell activation and antibody production against T-cell-dependent antigens.

The data on the importance of local Zn concentration in the liver and spleen for the normal function of mononuclear phagocytes and various subsets of lymphocytes (4,5) support our findings. According to them, zinc may directly induce monokine secretion by monocytes, but its effect on T cells appear to be contradictory and dose dependent, because T-cell

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stimulation by zinc takes place only in concentrations high enough for the monokine induction, but below the critical concentration for T-cell suppression (9). High zinc concentrations inhibit T-cell proliferation by blocking the interleukin-1 (IL-1) type I receptor-associated kinase (9). Zinc uptake studies revealed that within a few minutes, zinc may be taken up by peripheral blood mononuclear cells (PBMC), reaching nearly equal levels in PBMC for isolated monocytes and T cells, indicating that although zinc exerts different effects in monocytes and lymphocytes, zinc uptake does not differ between these cell types (9). In PBMC, zinc predominantly induces IL-1, IL-6, and tumor necrosis factor-0~ (TNF-~) and therefore has an immense immunoregulatory capacity (9). Furthermore, it was emphasized that zinc ions regulate the Thl/Th2 relationship and their specific cytokine production (10,11). Thus, deficiencies of zinc ions accelerated a shift of immune functions from a predominantly cellular Thl response to a humoral Th2 response (10) and decreased the production of interferon-7, IL-2, and TNF-o~ (11). The later data also emphasized that zinc may be required for the regeneration of new CD4 § T lymphocytes and maintenance of T cytolytic cells (11). In support to the role of the liver in these events, it was reported that long-term zinc supplementation in patients with chronic liver disease, which have reduced serum levels of zinc, may stimulate DNA synthesis and cytokine production by PWM-stimulated PBMC, and normalize the elevated levels of IL-6 and IL-10 in serum (12).

In addition to these data showing the direct role of Zn for the proliferation and function of several lymphatic cells, the central role of the liver in the metallothionein metabolism must be emphasized. As is well known, MT is able to regulate the plasmatic Zn concentration and redis- tribution of zinc to other organs (2). Reduction in plasma Zn concentration is usually associated with enhanced hepatic MT gene expression and enhanced synthesis of MT (13). Owing to the greater binding activity of zinc to the hepatic MT than to the main thymic hormon-thymulin (for review, see ref. 14), it was hypothesized that an increment of hepatic MT may simultaneously decrease the formation of biologically active thymulin (15), which requires zinc in an equimolar ratio for full activity (16). This hypothesis probably explains our data showing that during the time of maximal immune response (on the fourth day in the PFC test, and on the seventh day in local HVGR; Figs. 3 and 5, respectively), linear regression lines have opposite directions in the liver and in the thymus. How- ever, the inverse relationship between the zinc concentration in the thymus and the intensity of immune response might also mean that thymic Zn had been utilized for the zinc-thymulin complex and was secreted from the thymus or that Zn was consumed during the commit- ment of lymphocytes to the T-cell lineage. It is notable that zinc-thymulin complexes may potentiate IL-1 activation of PKC in isolated splenic nuclei and promote the IL-2R expression on lymFhocytes (17).



In addition to these data implying the specific relationship between the hepatic, splenic, and thymic zinc concentration during the initial and central type of immune reaction, our data show that during the final phase of HVGR, a highly significant negative correlation (r = -8.593) between the intensity of local immune reaction and zinc concentration in the liver might be found (Fig. 5, tenth day). It is likely that this reflects the changes in hepatic MT concentrations, probably linked with the nor- malization of neuroendocrine homeostasis after the inoculation of an antigen, or the role of the Zn in processes of apoptosis at the end of an immune reaction, but the proofs are lacking. This hypothesis is, however, supported by published evidence showing that glucocorticoid hormones, glucagon and epinephrin may exert both positive (18) and negative influ- ences (19) on hepatic MT synthesis and by reports which emphasize a special relationship among the Zn deficiency, suppression of lymphatic tissue, and apoptosis (20).

In conclusion, regardless of unclear mechanisms of their induction, our data show that during the normal immune response, the significant changes occur in the endogenous Zn tissue concentration. In the begin- ning of local HVGR (on the fifth day after inoculation of semiallogeneic splenocytes) and during the maximal intensity of humoral immune reaction (fourth day after the sensitization with SRBC), a positive correlation between the hepatic and splenic as well as a negative correlation between thymic Zn concentration (fourth or seventh day) and intensity of immune reaction were found. On the contrary, in the final phase of local HVGR, the highly negative correlation between the hepatic Zn concentration and local increase in the weight of popliteal lymph nodes was observed.

The data not only emphasize the necessity of Zn for the development of normal immune response but also point to the to the existence of a Zn-dependent hepatothymic axis during the humoral and cellular types of immune reactivity in mice.

ACKNOWLEDGMENT

This work was supported by the grants from the Croatian Ministry of Science (projects No. 62003 and 62002).

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3. P. J. Fraker, M. E. Gershwin, R. A. Good, and A. Prasad, Interrelationships between zinc and immune function, Fed. Proc. 45, 1474-1479 (1986).

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Tissue zinc dynamics during the immune reaction in mice

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