Biological Trace Element Research

, Volume 151, Issue 2, pp 234–239

Changes of the Serum Cytokine Contents in Broilers Fed on Diets Supplemented with Nickel Chloride

Authors

  • Bangyuan Wu
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
    • College of Veterinary MedicineSichuan Agricultural University
  • Xi Peng
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
  • Jing Fang
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
  • Zhicai Zuo
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
  • Jianying Huang
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
  • Qin Luo
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
  • Yubing Deng
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
  • Hesong Wang
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
  • Juan Liu
    • Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary MedicineSichuan Agricultural University
Article

DOI: 10.1007/s12011-012-9554-y

Cite this article as:
Wu, B., Cui, H., Peng, X. et al. Biol Trace Elem Res (2013) 151: 234. doi:10.1007/s12011-012-9554-y

Abstract

Cytokines are immunoregulatory proteins which play an important role in the immune system. The purpose of this study was to examine the serum cytokine contents including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) induced by dietary nickel chloride in broilers by enzyme-linked immunospecific assay. A total of 240 one-day-old avian broilers were divided into four groups and fed on a corn–soybean basal diet as control diet or the same basal diet supplemented with 300, 600, and 900 mg/kg of nickel chloride. During the experimental period of 42 days, the results showed that the serum IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α contents were lower (p < 0.05 or p < 0.01) in the 300, 600, 900 mg/kg groups than those in the control group. It was concluded that dietary nickel chloride in the range of 300 to 900 mg/kg could reduce the serum cytokine contents, which could finally impact the immune function in broilers.

Keywords

Nickel chlorideIL-2IL-4IL-6IL-10IFN-γTNF-αSerumBroiler

Introduction

The essentiality of nickel in the nutrition of people (especially for children) and different classes of livestock has been demonstrated [13]. Anke et al. [4] reported an absolute requirement for nickel in the ruminants (sheep, goat, and so on). Some other evidence is also reviewed that nickel is an essential element for the chick, rat, and pig. Nickel is required for activity of vitamin B12 and biotin during metabolism of odd-chain fatty acid in animals [5], and is involved in methionine-folate metabolism [68]. Interactions between nickel and copper, zinc, iron, calcium, and some other trace minerals in the body have also been reported [911]. And during the past decade, nickel has been shown to participate in several enzyme reactions. In chemoautotrophic hydrogen bacteria, nickel is incorporated into hydrogenases [12], and carbon monoxide dehydrogenase from clostridia has also been shown to be a nickel enzyme [1316]. Urease was the first enzyme shown to contain nickel [17]. And there is evidence that some essential trace elements including nickel may also be of nutritional significance under stress conditions [18].

But during the last decade, as a result of industrial development, the environmental pollution is becoming a serious problem that should receive careful attention in the world. Lots of chemical substances have generated pollution in air, water, and soil. Nickel is a ubiquitous trace metal and occurs in soil, water, air, and in the biosphere, which is emitted into the environment from both natural and man-made sources [19, 20]. Nickel, high concentrations of which can affect human health badly, can accumulate on plants, animals, and soil [21].

Nickel is present in most of the dietary items, and food is considered to be a major source of nickel exposure for the general population. Nickel content in food may considerably vary from place to place due to the difference in nickel content of the soil. However, certain foods (oatmeal, dried beans and peas, nuts, dark chocolate and soya products) are routinely high in nickel content [22]. Nickel in the diet of a nickel-sensitive person can provoke dermatitis. This can influence the outcome of the disease and can benefit the nickel-sensitive patient [23]. Also, nickel compounds have been found to exhibit carcinogenic properties based on epidemiological, experimental animal and cell culture studies. Exposure to soluble nickel compound produces toxic effects on immune system, but the mechanism of action remains to be elucidated. Oral administration of nickel sulfate for 35 days to mice/rats has been shown to decrease reproductive tissue weights, sperm count and motility [24, 25].

There are some studies on the effect of nickel sulfate on synthesis of the serum interleukin-2 (IL-2), interleukin-5 (IL-5), tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) in human. However, no systematic studies have been reported about the changes of the serum cytokine contents induced by dietary nickel chloride in animals so far. In the present study, the broilers fed on diets supplemented with nickel chloride were used to investigate the changes of the serum IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ contents in broilers by the method of enzyme-linked immunosorbent assay (ELISA), which provided a new experimental evidence for clarifying the mechanisms of the effect of nickel chloride on the creation or secretion of the cytokines and at last evaluated the immune function in broilers.

Materials and Methods

Chickens and Diets

Two hundred forty 1-day-old healthy avian broilers (purchased from a commercial rearing farm of Wenjiang poultry farm in Sichuan province) were randomly were divided into four groups with 60 broilers in each group. Broilers were housed cages with electrically heated units and were provided with water as well as the undermentioned experimental diets ad libitum for 42 days.

A corn–soybean basal diet formulated by the National Research Council [26] was the control diet. Nickel chloride (NiCI2·6H2O) was mixed into the corn–soybean basal diet to produce experimental diets with 300, 600, 900 mg/kg of nickel chloride, respectively.

Sample Preparation

Five broilers in each group were phlebotomized from jugular vein to obtain serum at 14, 28, and 42 days of age during the experiment. Blood was clotted for 15 min and then centrifugated for 15 min at the speed of 3,000 rpm. The serum was removed and assayed immediately.

Determination of the Serum Cytokines Contents

Contents of the serum cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α) were determined using ELISA as described by Gaca et al. [27]. Those cytokine contents in the serum were quantified using the IL-2 (REF:DZE40115; LOT:201207), IL-4 (REF:DZE40018; LOT:201207), IL-6 (REF:DZE40017; LOT:201207), IL-10(REF:DZE40022; LOT:201207), IFN-γ (REF:DZE40046; LOT:201207), and TNF-α (REF:DZE40041; LOT:201207) ELISA kits specific for chick (RD Ltd, USA). The IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α contents were determined by the standard curve and expressed as nanogram per liter or pictogram per milliliter.

Methods

  1. 1.

    Prepare standard dilution by adding 50 μL per well of appropriate dilution to the plate. To do this, perform fivefold serial dilutions of the top standard either within the plate or in separate tubes. To dilute within the plate, add 100 μl chicken IL or IFN or TNF standard and 50 μl standard diluent to the first and second well and mix with together. Perform serial dilution by taking 100 μl standard from the first and second well to the third and the fourth well and continue for each subsequent well to the tenth. Therefore, the final chicken IL or IFN or TNF standard concentrations will be 60, 40, 20, 10, 5 ng/L. Standard diluent alone serves as the zero standards for row A of first to tenth well. For test samples, dilute fivefold with sample diluent and add 10 μL/well. Incubate at room temperature for 30 min.

     
  2. 2.

    Wash entire plate four times with 1× wash buffer.

     
  3. 3.

    Add 50 μL HRP–conjugate reagent to each well (except blank well) and incubate at room temperature for 30 min.

     
  4. 4.

    Wash plate six times with 1× wash buffer. For this final wash, soak wells in wash buffer for 10 s for each wash. This will help minimize background.

     
  5. 5.

    Add 50 μL of chromogen solution A and 50 μL of chromogen solution B to each well. Incubate at room temperature for 15 min.

     
  6. 6.

    Add 50 μL of stop solution to each well. The color in the wells should change from blue to yellow. If the color in the wells turns green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

     
  7. 7.

    Determine the optical density of each well within 30 min using a microplate reader set to 450 nm.

     

Calculate

The standard curve was drawn according to the standard density as the vertical. Then, the corresponding density was found out in the standard curve according to the sample OD value. The IL or IFN OR TNF contents were corresponding density multiplied by the dilution multiple.

Statistical Analysis

The significance of difference among 4 groups was analyzed by variance analysis, and results presented as means ± standard deviation (\( \overline{X}\pm \mathrm{S} \)). The analysis was done under SPSS 12.0 for windows. A value of p < 0.05 was considered significant.

Results

Changes of the Serum IL-2 Content

The results were showed in Table 1. The serum IL-2 contents were significantly lower (p < 0.01) in the 300, 600, and 900 mg/kg groups than those in the control group from 14 to 42 days of age.
Table 1

Change of the serum IL-2 content in the broiler

Group

IL-2 content (ng/L)

14 days

28 days

42 days

Control group

276.16 ± 8.41

317.27 ± 8.93

319.32 ± 6.68

300 mg/kg group

244.10 ± 8.84**

294.40 ± 8.35**

290.00 ± 4.90**

600 mg/kg group

250.68 ± 8.07**

232.21 ± 9.55**

234.12 ± 9.41**

900 mg/kg group

211.15 ± 2.45**

180.67 ± 8.85**

180.06 ± 8.99**

Data are presented with the means ± standard deviation (n = 5)

*p < 0.05, compared with the control group

**p < 0.01, compared with the control group

Changes of the Serum IL-4 Content

The serum IL-4 contents were significantly decreased in the group II and group III at 14 days of age, and were significantly lower in the 300, 600, and 900 mg/kg groups than those in the control group at 28 and 42 days of age. The results were showed in Table 2.
Table 2

Change of the serum IL-4 content in the broiler

Group

IL-4 content (ng/L)

14 days

28 days

42 days

Control group

235.95 ± 5.09

203.94 ± 4.90

202.09 ± 5.15

300 mg/kg group

228.42 ± 6.08

191.20 ± 5.21**

181.96 ± 7.97**

600 mg/kg group

202.82 ± 6.84**

169.73 ± 5.59**

151.42 ± 4.67**

900 mg/kg group

1 78.90 ± 7.57**

151.69 ± 5.51**

125.05 ± 5.97**

Data are presented with the means ± standard deviation (n = 5)

*p < 0.05, compared with the control group

**p < 0.01, compared with the control group

Changes of the Serum IL-6 Content

The serum IL-6 contents were significantly reduced (p < 0.01) in the 300, 600, and 900 mg/kg groups from 14 to 42 days of age when compared with those of the control group except the 300 mg/kg group at 28 days of age, which was showed in Table 3.
Table 3

Change of the serum IL-6 content in the broiler

Group

IL-6 content (ng/L)

14 days

28 days

42 days

Control group

363.02 ± 5.51

344.29 ± 2.02

344.73 ± 3.06

300 mg/kg group

344.12 ± 3.82**

339.63 ± 5.04

335.00 ± 2.90**

600 mg/kg group

325.13 ± 2.87**

305.00 ± 3.40**

316.61 ± 3.37**

900 mg/kg group

254.92 ± 3.18**

287.15 ± 2.80**

264.19 ± 2.81**

Data are presented with the means ± standard deviation (n = 5)

*p < 0.05, compared with the control group

**p < 0.01, compared with the control group

Changes of the Serum IL-10 Content

As showed in Table 4, the serum IL-10 contents were significantly reduced (p < 0.01) in the 600 and 900 mg/kg groups at 14 days of age and were significantly lower (p < 0.01) in the 300, 600, and 900 mg/kg groups than those in the control group from 28 to 42 days.
Table 4

Change of the serum IL-10 content in the broiler

Group

IL-10 content (pg/mL)

14 days

28 days

42 days

Control group

284.96 ± 6.89

248.14 ± 8.67

338.40 ± 6.63

300 mg/kg group

276.63 ± 9.86

219.17 ± 7.75**

294.87 ± 5.60**

600 mg/kg group

247.96 ± 7.54**

210.34 ± 6.72**

226.671 ± 8.71**

900 mg/kg group

245.08 ± 7.37**

193.31 ± 5.51**

168.43 ± 8.84**

Data are presented with the means ± standard deviation (n = 5)

*p < 0.05, compared with the control group

**p < 0.01, compared with the control group

Changes of the Serum TNF-α Content

The TNF-α contents were reduced (p < 0.05 or p < 0.01) in the 300, 600, and 900 mg/kg groups from 14 to 42 days of age when compared with those of control group. The results were showed in Table 5.
Table 5

Change of the serum TNF-α content in the broiler

Group

TNF-α content (pg/mL)

14 days

28 days

42 days

Control group

633.43 ± 13.88

602.56 ± 12.68

666.14 ± 16.66

300 mg/kg group

610.12 ± 11.15*

586.68 ± 9.71**

580.82 ± 19.01**

600 mg/kg group

578.86 ± 9.33**

527.66 ± 15.17**

483.36 ± 14.30**

900 mg/kg group

432.49 ± 11.27**

493.26 ± 12.98**

457.67 ± 15.36**

Data are presented with the means ± standard deviation (n = 5)

*p < 0.05, compared with the control group

**p < 0.01, compared with the control group

Changes of the Serum IFN-γ Content

The results in Table 6 showed that the serum IFN-γ contents were markedly decreased (p < 0.01) in the 300, 600, and 900 mg/kg groups from 14 to 42 days of age in comparison with those of the control group.
Table 6

Change of the serum IFN-γ content in the broiler

Group

IFN-γ content (ng/L)

14 days

28 days

42 days

Control group

516.38 ± 6.74

523.93 ± 8.70

494.44 ± 5.94

300 mg/kg group

500.20 ± 4.88**

496.94 ± 4.33**

458.78 ± 7.03**

600 mg/kg group

490.56 ± 3.01**

466.34 ± 8.91**

384.84 ± 9.05**

900 mg/kg group

457.35 ± 4.94**

407.94 ± 5.56**

360.12 ± 8.13**

Data are presented with the means ± standard deviation (n = 5)

*p < 0.05, compared with the control group

**p < 0.01, compared with the control group

Discussion

The immune system can be divided into an innate part and adaptive part. The former consists mainly of monocytes, natural killer (NK), and dendritic cells (DC), and the latter is represented by B and T lymphocytes [28]. Innate immunity represents the first line of host defense and provides the basis for an adequate response to pathogens. Besides phagocytic cells (neutrophils, monocytes, macrophages, and DCs) and NKs, soluble mediators such as cytokines, chemokines, hormones, and/or oxygen-free radicals are also of importance within the innate immune system [29]. The adaptive immune system generates responses involving T and B lymphocytes together with specific antibodies and a range of cytokines and chemokines. It has become clear that these two systems function cooperatively to provide the optimal host defense, and a defect in either system can have a significant adverse effect on immune functions [30]. Therefore, the determination of the number of T or B cells, the quantitative or qualitative measure of the cytokines and antibody levels, or the study of cellular function such as phagocytic activity is used to evaluate the state of the immune system [31].

Boscolo et al. [32] have reported that nickel sulfate can induce IL-2 and IL-5 synthesis in women serum, and Salsano et al. [33] have reported that nickel sulfate can induce IFN-γ and TNF-α synthesis, which illustrates that nickel can affect the synthesis and secretion of cytokines. In the present study, we found that the serum IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ contents in broilers were decreased in the nickel chloride groups, implying that nickel chloride could also inhibit the synthesis or secretion of the abovementioned cytokines as nickel sulfate.

It is well known that T lymphocytes, which are required for both cell-mediated immune responses and the production of antibody by B lymphocytes, are composed of two distinct subsets—T helper 1 (Th1) and T helper 2 (Th2) cells. Th1 cells produce IL-2, IFN-γ, and TNF and contribute to cytotoxic T cell differentiation, mediate cellular immune responses, etc., whereas Th2 cells produce IL-4, IL-5, IL-6, and IL-13, promote B cell proliferation and differentiation, and mediate humoral immune response [34, 35].

The source of cytokines produced in various conditions has been investigated in numerous studies. IL-2 is secreted by activated T cells, and there is a close relationship between IL-2 and maturity, proliferation of T cells [36]. IL-4, also called B lymphocytes growth factor-1 (BCGF-1), can promote mast cell, B and T lymphocyte proliferation, differentiation and the process of forming SIgA [37] and plays an important role in the immune system [38]. IL-6, which was originally identified as a B lymphocyte differentiation factor, is now known to be a multifunctional cytokine that regulates the immune response, hematopoiesis and inflammation. Many clinical data and animal models suggest that IL-6 plays critical roles in the pathogenesis of autoimmune diseases [39]. IL-6 and IL-10 are produced by a variety of cells, including monocytes and T cells, and can exert its effects on both myeloid and lymphoid cells [40]. However, in our study, the serum IL-2, IL-4, IL-6, and IL-10 contents were reduced in the nickel chloride groups, indicating that the proliferation, differentiation, maturity and activation of B and T lymphocytes were decreased by dietary nickel chloride. And the immune function was finally impaired in broilers.

TNF-α, secreted by activated T cells, is a potent pro-inflammatory and immunomodulatory cytokine implicated in inflammatory conditions. The cytokine IFN-γ is originally identified as an antiviral factor and plays central roles in the activation of macrophages, stimulation of antigen presentation through class I and class II major histocompatibility complex molecules [41], and regulation of T cell differentiation [42]. IFN-γ can also stimulate expression of other cytokines that activate and induce proliferation of CD4+ cells [43]. Thus, IFN-γ makes a major contribution to the cell-mediated immune response [44]. IL-2, TNF-α, and IFN-γ are produced by Th1 lymphocytes, which enhance cell-mediated immunity. The effect of the predominant Th1 results in activation of T lymphocytes, particularly cytotoxic functions and macrophages. However, both TNF-α and IFN-γ contents in the serum were decreased in the nickel chloride groups in the present study, indicating that the cell-mediated immune response was impaired by nickel chloride.

According to the results of the present study and the aforementioned discussion, it is concluded that dietary nickel chloride in the range of 300 to 900 mg/kg can reduce the serum IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ contents in broilers. The immune function can be finally impacted by decrease in lymphocyte numbers or lymphocyte activation.

Acknowledgments

The study was supported by the program for Changjiang scholars and innovative research team in university (IRT 0848) and the Education Department and Scientific department of Sichuan Province (09ZZ017).

Copyright information

© Springer Science+Business Media New York 2012