Experimental immunology
The proliferation rate and phase growth of human peripheral lymphocytes in vitro under zinc influence

Introduction

Zinc (Zn) is a trace element essential to normal growth of all living organisms. Human body contains around 2-4 γ of Zn, but its physiological concentration in plasma is low and tightly regulate in 12-16 M/L range. In human, serum zinc is protein bound: albumin (60%), transferrin (10%) and 2-macroglobulin (30%). Zinc is cofactor of over 300 enzymes, participates in cell processes such as signal transduction, transcription and replication [1-6].

Zinc is also necessary to proper immune system functioning. Zinc deficiency can lead to lower activity of immune system cells including T-lymphocytes, NK cells, macrophages and neutrophiles; it also decrease production of cytokines and affects apoptosis [1, 2, 5, 7, 8]. It has been shown that zinc deficiency decreases proliferation of lymphocytes after mitogen stimulation [4, 9, 10].

However, it was observed that zinc excess can be harmful causing similar symptoms to its deficiency – including inhibition of lymphocytes proliferation [1-3].

The aim of the following study was to examine the effect of different zinc chloride (ZnCl2) concentrations on peripheral blood lymphocytes proliferation in vitro. It was also verified which phase of lymphocytes proliferation is affected by ZnCl2.

Material and methods

Heparinized samples of blood (8 ml) were collected from healthy donors at Regionalne Centrum Krwiodawstwa i Krwiolecznictwa in Poznań, Poland. Gentamycine (Sigma Chemicals, St. Louis, USA) was added as a preservation to Eagle’s liquid (Biomed, Poland). Medium was later used to isolate lymphocytes and culture growing.

In order to isolate lymphocytes from blood it was mixed with Eagle’s medium in 1 : 1 ratio and centrifugated (25 minutes, 1750 RPM, temp. 5°C) over 5 ml of Gradisol-L (Aqua-Med, Poland). Cells were washed twice. The lymphocyte suspension (25 × 104 cells/ml) in Eagle’s medium was supplemented with 10% fetal bovine serum (Sigma Chemicals, St. Louis, USA). To stimulate proliferation phytohaemagglutinin-L (PHA-L, Roche Diagnostics, Sweden) was used in a concentration of 2.5 g/ml. Cultures were incubated with CO2 incubator under controlled conditions (5% CO2, temp. 37°C, humi-dity 95%).

In the first part of experiment after 48 h of incubation ZnCl2 (POCH SA) was added to the culture in four different concentrations (1 M, 10 M, 50 M and 100 M). Simultaneously [3H]-thymidine (Amersham, UK) was added in 1 Ci/well concentration and incubated for next 24 h.

Second part of experiment investigated which phase of lymphocytes proliferation is affected by ZnCl2. Cultures were prepared as above, two concentration of ZnCl2 were added (50 M, 100 M) in four time intervals: at the beginning of culture (0 h), after 4 h, 24 h and 48 h of incubation.

Twenty attempts were conducted in every part of the experiment.

Following samples marks were used: C – control; 1 – 1 M ZnCl2; 10 – 10 M ZnCl2; 50 – 50 M ZnCl2; 100 – 100 M ZnCl2.

In order to measure lymphocytes proliferation, cultures were transferred by the harvester (SKATRON Instruments, Norway) on glass fiber filters (Perkin Elmer, Massachusetts, USA), later placed in a scintillation cocktail. Measurement of thymidine incorporation was determined using scintillation counter (Perkin Elmer, Massachusetts, USA). Results were expressed in counts per minute (CPM).

Statistic analysis were determined by Statistica 8.0 software (StatSoft, USA). Wilcoxon signed-rank test was used to compare control and ZnCl2 affected samples. P va­lue < 0.05 was considered as statistically significant.

Results

Results indicates that effect of zinc chloride on lymphocyte proliferation after 48 h is determined by its concentration. Mean thymidine incorporation in cultures affected by ZnCl2 was lower than mean thymidine incorporation in the control cultures (free of ZnCl2) only for 100 M ZnCl2 concentration (Fig. 1). Difference was statistically significant (Wilcoxon test, p < 0.05).

Effect of 1 M, 10 M and 50 M zinc chloride concentrations on investigated process were inconsiderable and statistically insignificant.

In the second part of experiment relation between time of ZnCl2 addition and lymphocyte proliferation was demonstrated. The sooner zinc chloride was added, the stronger inhibition of thymidine incorporation was observed; the greatest change was given by 100 M concentration of ZnCl2 (Fig. 2). Statistically significant differences between control and zinc-affected samples (50 M and 100 M) were observed when zinc was added at the beginning (0 h) and after 4 hours of the cell culture. Addition of ZnCl2 after 48 h of cell culturing resulted in statistically significant difference with control sample only for 100 M concentration (Wilcoxon test, p < 0.01).

Samples with ZnCl2 added at the start of the cell culture have shown a decrease of mean thymidine incorporation to 84% (50 µM) and 59% (100 µM) in comparison to control. In subsequent time intervals following values were observed: 4 h: 90% (50 µM) and 72% (100 µM); 24 h: 102% (50 µM) and 93% (100 µM) and 48 h: 102% (50 µM) and 81% (100 µM).

Discussion

In our research the effect of zinc on lymphocytes proliferation was determined by its concentration in cell culture. Only 100 µM ZnCl2 concentration significantly inhibited tested process.

As mentioned before, zinc excess, as well as its deficiency, can negatively affect immune system, including lymphocytes proliferation. Moreover, zinc can stimulate or inhibit the same process depending on its concentration [1]. Ryu et al. who studied thymidine incorporation in mouse embryonic stem cells observed that low (40 M) concentrations of ZnCl2 stimulates the process, while higher doses ( 200 M) blocks the proliferative effect [11].

High zinc concentrations, exceeding physiological concentration 7-8 times (circa 100 µM) can inhibit lymphocytes functions and its proliferation [1-3].

Inhibition of lymphocytes proliferation by high concentrations of Zn was also observed by others [12-14]. Chang et al. demonstrated that physiologic concentrations of zinc has no effect on human peripheral blood mono-nuclear cells, higher doses (100 M) decreased cell proliferation and induced apoptosis [12]. Wellinghausen et al. [13] suggests that observed inhibition effect is connected with inhibition of type-1 receptor for IL-1 associated with protein kinase (IRAK) and decrease of signal transduction at very early stage. It was also observed that 100 M concentration stimulates monocytes, but inhibits T-lymphocytes activation due to its lower zinc content in cells, resulting in higher sensitivity. Concentration higher 3-4 times (60 µM) than physiological doesn’t lead to T-lymphocytes suppression but inhibits alloreactivity of mixed lymphocytes cultures MLC [14].

Viability of normal human lymphocytes and human myelogenous leukemia K562 cells exposed to different zinc concentrations was also studies [15]. Zinc evoked a concentration-dependent decrease in the viability of both types of cells, but normal lymphocytes much better survived zinc treatment than the cancer cells. Also some of the latest studies indicates that high concentrations of zinc can inhibit proliferation of some cancer cells such as Eca 109 [16].

In our research inhibition of lymphocytes proliferation was most significant for samples affected by ZnCl2 at the beginning of in vitro cultures (0 h). It means that high concentrations of zinc have negative effect on early phases of signal transduction after mitogen stimulation, what corresponds with Wellinghausen et al. [14] research.

References

 1. Haase H, Rink L (2009): The immune system and the impact of zinc during aging. Immun Ageing 6: 9.  

2. Ibs KH, Rink L (2003): Zinc-altered immune function. J Nutr 133 (5 Suppl 1): 1452-1456.  

3. Rink L, Gabriel P (2000): Zinc and the immune system. Proc Nutr Soc 59: 541-552.  

4. Rink L, Kirchner H (2000): Zinc-altered immune function and cytokine production. J Nutr 130 (5 Suppl): 1407-1411.  

5. Wellinghausen N, Rink L (1998): The significance of zinc for leukocyte biology. J Leukoc Biol 64: 571-577.  

6. Auld DS (2001): Zinc coordination sphere in biochemical zinc sites. Biometals 14: 271-313.  

7. Bao B, Prasad A, Beck FW, et al. (2006): Toxic effect of zinc on NF-B, IL-2, IL-2 receptor  and TNF- in HUT-78 (Th0) cells. Toxicol Lett 166: 222-228.  

8. Rink L, Haase H (2007): Zinc homeostasis and immunity. Trends Immunol 28: 1-4.  

9. Duchateau J, Delespesse G, Vereecke P (1981): Influence of oral zinc supplementation on the lymphocyte response to mitogens of normal subjects. Am J Clin Nutr 34: 88-93.

10. Klecha AJ, Salgueiro J, Wald M, et al. (2005): In vivo iron and zinc deficiency diminished T- and B-selective mitogen stimulation of murine lymphoid cells through protein kinase C-mediated mechanism. Biol Trace Elem Res 104: 173-183.

11. Ryu JM, Lee MY, Yun SP, Han HJ (2009): Zinc chloride stimulates DNA synthesis of mouse embryonic stem cells: involvement of PI3K/Akt, MAPKs, and mTOR. J Cell Physiol 218: 558-567.

12. Chang KL, Hung TC, Hsieh BS, et al (2006): Zinc at pharmacologic concentrations affects cytokine expression and induces apoptosis of human peripheral blood mononuclear cells. Nutrition 22: 465-474.

13. Wellinghausen N, Martin M, Rink L (1997): Zinc inhibits interleukin-1-dependts T cell stimulation. Eur J Immunol 27: 2529-2535.

14. Campo CA, Wellinghausen N, Faber C, et al. (2001): Zinc inhibits the mixed lymphocyte culture. Biol Trace Elem Res 79: 15-22.

15. Sliwinski T, Czechowska A, Kolodziejczak M, et al. (2009): Zinc salts differentially modulate DNA damage in normal and cancer cells. Cell Biol Int 33: 542-547.

16. Xiao HJ, Huang CY, Wang HY, Li M (2008): Effect of selenium and zinc on the proliferation of human esophageal cancer Eca109 cell line in vitro. Nan Fang Yi Ke Da Xue Xue Bao 28: 2117-2120.