Immunomodulatory effects of lactate dehydrogenase in vitro and in vivo

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Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/jff

Immunomodulatory effects of lactate dehydrogenase in vitro and in vivo Miho Daifukua, Kosuke Nishia, Takeaki Okamotob, Sogo Nishimotoc, Takuya Sugaharaa,d,* a

Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, Japan Faculty of Education, Ehime University, Matsuyama, Ehime 790-8577, Japan c Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama, Ehime 790-8577, Japan d South Ehime Fisheries Research Center, Ehime University, Ainan, Ehime 798-4292, Japan b

A R T I C L E I N F O

A B S T R A C T

Article history:

Lactate dehydrogenase (LDH) has been demonstrated to have an immunoglobulin produc-

Received 17 April 2012

tion-stimulating effect on an IgM-producing human hybridoma cell line and on human

Received in revised form

peripheral blood lymphocytes in vitro. Here, we examined the immunostimulatory activity

24 July 2012

of LDH on mouse immune system in vitro and in vivo. LDH stimulated IgA, IgG, and IgM pro-

Accepted 26 July 2012

duction by lymphocytes from spleen, mesenteric lymph node, and Peyer’s patch in vitro.

Available online 24 August 2012

BALB/c mice were administered with LDH at 1.0 or 5.0 mg/kg/day for 2 weeks to examine

Keywords:

by administration at 5.0 mg/kg/day. Moreover, IgA and IgG production by Peyer’s patch

Immunoglobulin

lymphocytes from mice administered at 1.0 mg/kg/day of LDH was accelerated. IgA, IL-4,

Immunomodulatory activity

IL-5, IL-10, IFN-c, and TNF-a production by spleen lymphocytes were also facilitated by

the immunostimulatory activity of LDH in vivo. The IgA level in mouse serum was increased

Lactate dehydrogenase

administration of LDH. It was revealed that oral administration of LDH stimulates immunoglobulin and cytokine production by lymphocytes in vivo.  2012 Elsevier Ltd. All rights reserved.

1.

Introduction

Functional food is defined as a food that satisfactorily demonstrates beneficial effects on one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either improved state of health and well-being and/or reduction of disease risk (Diplock et al., 1999). Functional foods are expected to improve physical function and to reduce the risk of specific pathologies by modulating im-

mune, secretion, nerve, circulating, and/or digestive systems. For example, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are well known as functional substances. DHA plays major roles in multiple signaling cascades contributing to cell growth, cell differentiation, function, and repair of the nerve system (Bazan, 2003; Lamaziere et al., 2011; Laye´, 2010). It was also indicated that DHA and EPA inhibit the growth of cancer cells and inflammation (Heller et al., 2004; Khan et al., 2006).

* Corresponding author: Address: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan. Tel./fax: +81 89 946 9863. E-mail address: [email protected] (T. Sugahara). Abbreviations: ConA, concanavalin A; CSR, class switch recombination; DHA, docosahexaenoic acid; ELISA, enzyme-linked immunosorbent assay; EPA, eicosapentaenoic acid; FBS, fetal bovine serum; GALT, gut-associated lymphoid tissue; IFN, interferon; Ig, immunoglobulin; IL, interleukin; LDH, lactate dehydrogenase; MLN, mesenteric lymph node; NaPB, sodium phosphate buffer; PP, Peyer’s patches; SD, standard deviation; TGF, transforming growth factor; TNF, tumour necrosis factor 1756-4646/$ - see front matter  2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jff.2012.07.005

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Several food factors have been found that stimulate the immune system such as lysozyme in hen egg, proteose-peptone component 3 fragment in fermented bovine milk, collagen in jellyfish, and ribulose-1,5-bisphosphate carboxylase/ oxygenase in kale on human peripheral blood lymphocytes in vitro or on mice in vivo (Murakami, Sasaki, & Sugahara, 1997; Nishi et al., 2011; Sugahara et al., 2005, 2006). We recently revealed that a water-soluble extract from yellowtail hearts enhances immunoglobulin (Ig) production by mouse lymphocytes in vitro and in vivo (Daifuku et al., 2011). The active substance was purified and identified as tropomyosin. In addition, a water-soluble extract from tuna bulbus arteriosus stimulated Ig and cytokine production by mouse primary lymphocytes (Daifuku et al., 2012). Tuna bulbus arteriosus is an elasticity tissue located in exit of the ventricle and helps smooth blood flow through the ventricle. Lactate dehydrogenase (LDH), triosephosphate isomerase, enolase, and haemoglobin were determined as the active substances in the extract. These four proteins stimulated the IgM production by human hybridoma HB4C5 cells. LDH (EC 1.1.1.27) plays a crucial role in maintaining aerobic metabolism by converting lactate to pyruvic acid. Triosephosphate isomerase (EC 5.3.1.1) catalyzes the isomerization from D-glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. Enolase (EC 4.2.1.11) catalyses the dehydration of 2-phosphoglyceric acid to phosphoenolpyruvate. Haemoglobin works to transport oxygen. LDH, triosephosphate isomerase, and enolase are glycolysis enzymes that exist in muscle and synthesize ATP from glucose for energy production. We have previously revealed that LDH and enolase facilitated the Ig production by HB4C5 cells and human peripheral blood lymphocytes (Daifuku et al., 2012; Sugahara, Shimizu, Abiru, Matsuoka, & Sasaki, 1998; Takenouchi & Sugahara, 2003). The Ig production stimulatory activities of these two enzymes were irrespective of their enzymatic activities. In this study, we focused on the immunostimulatory activity of LDH in vivo.

2.

Materials and methods

2.1.

Reagents

RPMI 1640 medium, penicillin, and streptomycin were products of Sigma (St. Louis, MO, USA). LDH from rabbit muscle was purchased from Oriental Yeast (Tokyo, Japan). LDH was dialyzed against 10 mM sodium phosphate buffer (NaPB; pH 7.4) before use to remove ammonium sulphate and sterilized by filtration.

2.2.

Experimental animal

Five-week-old female BALB/c mice (body weight: 14–19 g) were purchased from Japan SLC (Shizuoka, Japan) and kept in a specific pathogen-free facility. They were given free access to food and water, and the animal room was maintained under controlled conditions of 12 h light/12 h dark cycle at 25 ± 1 C and 55 ± 5% of humidity. All animal experiments described herein were carried out in accordance with the protocol approved by the Laboratory Animal Care Committee of Ehime University (approval number: 08-U-2-1, validity date:

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31 March 2013). Mice were maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals of Ehime University.

2.3. Immunostimulatory lymphocytes in vitro

effects

of

LDH

on

mouse

After acclimatization to their housing environment for 7 days, 6-week-old female BALB/c mice were sacrificed by cervical dislocation, and the spleen, mesenteric lymph nodes (MLN), and Peyer’s patches (PP) were excised. A suspension of spleen lymphocytes was made by gently passing the minced organ through a cell strainer (BD Falcon, Franklin Lakes, NJ, USA) with a pore size of 40 lm. Spleen lymphocytes were hemolyzed twice with a lysis buffer (155 mM NH4Cl, 15 mM NaHCO3, 1 mM EDTA, pH 7.3), washed with phosphate-buffered saline, and suspended in RPMI 1640 medium supplemented with 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 5% fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA). MLN lymphocytes and PP lymphocytes were collected by the same procedure as described above for isolation of spleen lymphocytes. Lymphocytes prepared from the spleen, MLN, and PP were suspended in 5% FBS-RPMI 1640 medium supplemented with a various concentration of LDH and inoculated into each well of a 96-well cell culture plate (BD Falcon) at 1.0 · 106 cells/mL. After cultivation at 37 C for 24 h under humidified 5% CO295% air, culture supernatant was collected and the amounts of IgA, IgG, and IgM secreted into culture medium were determined by an in-house-developed enzyme-linked immunosorbent assay (ELISA) as described previously (Nishimoto et al., 2009). The assay was carried out in triplicate.

2.4.

Immunomodulatory effects of LDH in vivo

Following 1 week acclimation, 6-week-old female BALB/c mice were administered with LDH at 1.0 or 5.0 mg/kg/day for 14 days. Control mice were given 10 mM NaPB as a vehicle control. Blood was collected on day 15 and centrifuged at 18,000g for 20 min after leaving at room temperature for 1 h. Serum was then collected, and the amounts of Igs were measured by ELISA as described previously (Nishimoto et al., 2009). Collected lymphocytes derived from the spleen and PP were suspended in 5% FBS-RPMI 1640 medium and cultured in a 48-well cell culture plate. Spleen lymphocytes were inoculated at 1.0 · 106 cells/mL and cultured for 24 h to evaluate the Ig production or at 2.0 · 106 cells/mL and cultured for 48 h under stimulation with concanavalin A (ConA; Seikagaku, Tokyo, Japan) at 10 lg/mL to evaluate the cytokine production. PP lymphocytes were inoculated at 4.0 · 105 cells/mL and cultured for 48 h. The amounts of IgA, IgG, and IgM secreted into culture medium were measured by ELISA as described previously (Nishimoto et al., 2009). The amounts of interleukin (IL)-4, IL-5, IL-6, IL-10, tumour necrosis factor (TNF)-a, interferon (IFN)-c, and transforming growth factor (TGF)-b1 secreted into culture medium were measured by commercially available ELISA kits (eBioscience, San Diego, CA, USA). All ELISAs were done in triplicate.

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2.5.

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Statistical analysis

Results are expressed as the mean ± standard deviation (SD). Tukey’s test was used to assess the statistical significance of the difference against the control following analysis of variance. Each value of *p < 0.05, **p < 0.01, or ***p < 0.001 is considered to be statistically significant.

3.

Results

3.1. Immunomodulatory effect of LDH on mouse primary lymphocytes in vitro The Ig production-stimulatory effect of LDH on mouse spleen lymphocytes was examined. Mouse primary lymphocytes from the spleen were cultured in 5% FBS-RPMI 1640 medium supplemented with a various concentration of LDH. As summarized in Table 1, LDH stimulated IgA, IgG, and IgM production by spleen lymphocytes. Among them, IgA production was most enhanced and increased 2.5-fold at the protein concentration of LDH at 1.5 mg/mL compared with control. LDH also increased IgG and IgM production 1.6-fold and 1.5-fold compared to the control, respectively. Significant differences in Ig production were observed under these conditions. Next, an immunomodulatory effect of LDH on lymphocytes from MLN and PP was evaluated. Gut-associated lymphoid tissue (GALT) including MLN and PP is one of segments in mucosa-associated lymphoid tissue, which has a general immune architecture resembling that of systemic lymphoid tissues (Chen & Cerutti, 2010). However, mucosaassociated lymphoid tissue has several unique, anatomic and cellular features. Since exogenous substances can affect directly the mucosal surface, the gut immunity is open to influence of food components. Thus, we focused on the effect of LDH on lymphocytes from GALT and evaluated the immunomodulating activity of LDH on MLN lymphocytes and PP lymphocytes in vitro. It was found that LDH enhanced IgA, IgG, and IgM production by MLN lymphocytes (Fig. 1) and by PP lymphocytes (Fig. 2) in a dose-dependent manner, suggesting that LDH may affect both MLN and PP lymphocytes and modulate the gut immunity. The IgA, IgG, and IgM production by MLN lymphocytes was enhanced 2.5-, 2.1-, and 2.2-fold compared with the control, respectively. In the case of PP lymphocytes, IgA, IgG, and IgM production was enhanced 1.9-fold, 1.5-fold, and 1.7-fold compared with control, respectively. IgG and IgM production was more stimulated by both

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MLN and PP lymphocytes than by spleen lymphocytes. It is conceivable that LDH might affect lymphocytes in different ways depending on the derived tissue.

3.2.

Immunomodulating effect of LDH in vivo

Effects of LDH on mice in vivo were examined by oral administration of LDH. Six-week-old female BALB/c mice were administered with LDH at 5.0 or 1.0 mg/kg/day for 14 days. Doses were determined based on in vitro results and potential intake availability of LDH. Then, Ig concentrations in serum and in culture supernatant of lymphocytes derived from the spleen and PP were measured. IgA levels in serum increased in a dose-dependent manner and were significantly enhanced by administration at 5.0 mg/kg/day compared with the control group (Fig. 3). IgA production by spleen lymphocytes was significantly enhanced in the group administered at 5.0 mg/kg/day (Fig. 4). It was also confirmed that IgE production by spleen lymphocytes was not changed by administration of LDH (data not shown), suggesting that LDH does not induce allergy response. In PP lymphocytes, significant increases in IgA and IgM production were observed in the group administered at 1.0 mg/kg/day (Fig. 5). On the other hand, IgA and IgM production was not changed in the group administered at 5.0 mg/kg/day. In another administration experiment, it was confirmed that IgA, IgG, and IgM production by PP lymphocytes was significantly enhanced by administration of LDH at 3.0 mg/kg/day, an intermediate concentration between 1.0 and 5.0 mg/kg/day (data not shown). It is conceivable that an optimal administration concentration of LDH for lymphocytes from each tissue might exist. LDH was previously reported to lose its own enzymatic activity after digestion with trypsin (Takenouchi & Sugahara, 2003). On the other hand, partially digested LDH enhanced the IgM production-stimulating activity on human-human hybridoma HB4C5 cells and loss of the IgM production-stimulating activity was slower than that of its enzymatic activity. These facts indicated that the immunomodulating activity of LDH has little relation to its own enzymatic activity, and that LDH might keep the immunomodulating activity after gastrointestinal digestion at some level in vivo. Production of cytokines such as IL-4, IL-5, IL-10, IFN-c, and TNF-a by spleen lymphocytes under stimulation with ConA was significantly enhanced in the group administered with LDH at 5.0 mg/kg/day (Fig. 6). Although no significant difference was observed, IL-6 increased in a dose-dependent

Table 1 – Effect of LDH on Ig production by spleen lymphocytes. LDH (mg/mL)

IgA (ng/mL)

IgG (ng/mL)

IgM (ng/mL)

0 0.1 0.5 1.5

9.2 ± 1.5 14.5 ± 0.9*** 11.7 ± 0.4* 23.1 ± 1.0***

7.3 ± 1.1 8.9 ± 1.0 9.8 ± 1.5* 11.5 ± 0.3***

95 ± 13 127 ± 11* 127 ± 10* 140 ± 9*

Spleen lymphocytes were inoculated at 1.0·106 cells/mL in 5% FBS-RPMI 1640 medium supplemented with a various concentration of LDH and cultured for 24 h at 37 C under humidified 5% CO2-95% air. The amounts of IgA, IgG, and IgM produced by lymphocytes in each culture medium were measured by ELISA. Control represents Ig production by spleen lymphocytes cultured in 5% FBS-RPMI 1640 medium supplemented with 10 mM NaPB instead of LDH. Results are represented as the mean ± SD of three independent measurements. Statistically significant differences from control were represented as *p<0.05 or ***p<0.001.

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IgA

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IgG

IgM

14 0 14.0

400

60 6.0

12.0

200

5.0

10.0

Ig gM con nc. (ng//mL L)

Ig gG con nc. (ng/ ( /mL L)

Ig gA ccon nc. ((ng//mL L)

300

8.0 60 6.0 40 4.0

100

1

0.0

10 100 1000 10000 Protein conc conc. (µg/mL)

4.0

3.0 3 0

2.0

10 1.0

20 2.0 0

975

1

0 .0

10 100 1000 10000 Protein conc. conc (µg/mL)

1

10 100 1000 10000 Protein conc. conc (µg/mL)

Fig. 1 – Effect of LDH on Ig production by MLN lymphocytes in vitro. Mouse MLN lymphocytes were inoculated at 1.0 · 106 cells/ mL in 5% FBS-RPMI 1640 medium supplemented with various concentrations of LDH and cultured for 24 h at 37 C under humidified 5% CO2-95% air. The amounts of IgA, IgG, and IgM produced by lymphocytes in each culture medium were measured by ELISA. Control (d) represents Ig production by mouse MLN lymphocytes cultured in 5% FBS-RPMI 1640 medium supplemented with 10 mM NaPB instead of LDH. Results are represented as the mean ± SD of three independent measurements.

IgA

IgG

200

IgM 40 4.0

14 0 14.0 12.0

120

80

3 0 3.0

10.0

nc. ((ng//mL Ig gM con L)

( /mL Ig gG con c nc. (ng/ L)

Ig gA ccon nc. ((ng//mL L)

160

8.0 60 6.0

20 2.0

40 4.0 1.0

40 2 0 2.0 0 1

10 100 1000 10000 Protein conc conc. (µg/mL)

0.0 1

0.0 10 100 1000 10000 Protein conc. conc (µg/mL)

1

10 100 1000 10000 Protein conc. conc (µg/mL)

Fig. 2 – Effect of LDH on Ig production by PP lymphocytes in vitro. Mouse PP lymphocytes were inoculated at 2.0 · 106 cells/mL in 5% FBS-RPMI 1640 medium supplemented with various concentrations of LDH and cultured for 48 h at 37 C under humidified 5% CO2-95% air. The amounts of IgA, IgG, and IgM produced by lymphocytes in each culture medium were measured by ELISA. Control (d) represents Ig production by mouse PP lymphocytes cultured in 5% FBS-RPMI 1640 medium supplemented with 10 mM NaPB instead of LDH. Results are represented as the mean ± SD of three independent measurements.

manner. By contrast, TGF-b1 production by spleen lymphocytes decreased in a dose-dependent manner and declined significantly in the group administered at 5.0 mg/kg/day compared with control group. From these results, it was found that LDH affected not only Ig production but also cytokine production. Cytokines are produced by many sorts of lymphocytes and regulate the immune response, suggesting that LDH might enhance Ig production by affecting cytokine production.

4.

Discussion

Regarding the results of Ig production in vivo, LDH increased the IgA level in serum and the IgA productivity by lymphocytes from both the spleen and PP, indicating that LDH may particularly enhance IgA production. IgA is the predominant Ig isotype in intestinal secretions, and B cells residing in GALT mainly differentiate into IgA-producing plasma cells. The IgM+ B cells undergo class switch recombination (CSR) and

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Ig gA

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IgG

g IgM

25 2.5

25 2.5

07 0.7

* 06 0.6

Ig gG con c nc. (mg ( g/mL L)

Ig gA cconc. (mg ( /mL L)

15 1.5

1.0

Ig gM con c nc. (mg ( g/mL L)

20 2.0

20 2.0

15 1.5

1.0

0.5 0.4 03 0.3 02 0.2

0.5

0.5

0.1 0.0

Control

1 0 1.0

0.0

5 0 5.0

Control

mg/kg/day /k /d

1 0 1.0

0.0

5 0 5.0

Control

mg/kg/day /k /d

1 0 1.0

50 5.0

/k /d mg/kg/day

Fig. 3 – Effect of LDH on Ig levels in serum in vivo. Sera from mice administered with LDH at 5.0 mg/kg/day or 1.0 mg/kg/day for 14 days and were collected and the Ig levels was measured by ELISA. Each group consisted of 5 mice. A bar represents the average of Ig levels of 5 mice. Statistically significant differences from control were represented as *p < 0.05.

Ig gA

IgG

12 0 12.0 *

IgM g

80

160

60

120

80 8.0 6.0 40 4.0

Ig gM con nc. (ng/mL L)

Ig gG con nc. ((ng//mL L)

Ig gA con c nc. ((ng//mL L)

10.0

40

80

40

20 2.0

0.0

Control

1 1.0 0

5 5.0 0

0

Control

mg/kg/day

1.0 1 0

5.0 5 0

mg/kg/day

0

Control

1.0 1 0

5.0 5 0

mg/kg/day

Fig. 4 – Effect of LDH on Ig production by spleen lymphocytes ex vivo. Spleen lymphocytes were prepared from mice administered with LDH at 5.0 or 1.0 mg/kg/day for 14 days and incubated at 1.0 · 106 cells/ml in 5% FBS-RPMI 1640 medium for 24 h. The amounts of IgA, IgG, and IgM produced by lymphocytes were measured by ELISA. Each group consisted of 5 mice. A bar represents the average of Ig levels of 5 mice. Statistically significant differences from control were represented as *p < 0.05. differentiate to IgA+ B cells and eventually to IgA-producing plasma cells. There are mainly two ways to progress IgA CSR. One is the way called T cell-dependent CSR that needs activation of IgM+ B cells by CD4+ T cells expressing CD40 ligand and by TGF-b1 produced by Foxp3+ regulatory T cells, CXCR5+ T follicular helper cells, IL-10-producing regulatory1 cells, dendric cells, stromal cells, and B cells. Another way is T cell-independent CSR in which IgM+ B cells differentiate by the B cell-activating factor of TNF family and a proliferation-inducing ligand which are IgA CSR-inducing factors released by dendric cells, monocytes, neutrophils, basophils, and stromal cells. IgA-producing B cells migrate from the

inductive site of PP to the effector site of the gut lamina propria through blood circulation (Chen & Cerutti, 2010; Lamm & Phillips-Quagliata, 2002; Mora & von Andrian, 2008). Thus, it is possible that LDH promotes IgA CSR in PP and that classswitched IgA+ B cells circulated in whole body, leading to increases in IgA level in serum and in IgA production by lymphocytes from both the spleen and PP. Although TGF-b1 production by spleen lymphocytes was decreased by LDH administration, TGF-b1 production derived from GALT is more important in IgA CSR. In addition, IL-4, IL-5, and IL-10 production by spleen lymphocytes increased. These cytokines might relate to the increase in IgA production, because these

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Ig gA

IgG

300

IgM g 25 2.5

16 0 16.0 ***

250

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14 0 14.0 20 2.0

**

200

150

100

Ig gM con nc. ((ng//mL L)

Ig gG con nc. ((ng//mL L)

Ig gA ccon nc. (ng/ ( /mL L)

12 0 12.0 10 0 10.0 80 8.0 60 6.0 4.0

15 1.5

1.0

0.5

50 2.0 0

Control

1 1.0 0

5.0 50

0.0

0.0 Control

mg/kg/da mg/kg/day

1 0 1.0

5 0 5.0

Control

1 0 1.0

mg/kg/da mg/kg/day

5 0 5.0

mg/kg/da mg/kg/day

Fig. 5 – Effect of LDH on Ig production by PP lymphocytes ex vivo. PP lymphocytes were prepared from mice administered with LDH at 5.0 mg/kg/day or 1.0 mg/kg/day for 14 days and incubated at 4.0 · 105 cells/mL in 5% FBS-RPMI 1640 medium for 48 h. The amounts of IgA, IgG, and IgM produced by lymphocytes were measured by ELISA. Results are represented as the mean ± SD of 5 mice. Statistically significant differences from control were represented as **p < 0.01 or ***p < 0.001.

IL-5

IL-4 700

IL-6

80

*

*

250

5.0

300 200

40

IL-10 conc. (ng/mL)

400

60

IL- 6 conc. (pg/mL)

IL- 5 conc. (pg/mL)

IL- 4 conc. (pg/mL)

500

200 150 100

40 4.0 3.0 2.0

20 10 1.0

50

100 Control 1.0

0

5.0

Control 1.0

mg/kg/day /k /d

0

5.0

Control 1.0

mg/kg/day /k /d

IFN-γ

5.0

mg/kg/day /k /d

TNF-α *

1.4 1 4

0

Control 1.0

5.0

mg/kg/day /k /d

TGF-β

180

12 1.2

*

160

1.2

1.0

10 1.0 0.8 0.6 0.4

140

TGF-β conc. (ng/mL)

TNF-α conc. (pg/mL)

IFN-γ conc. (ng/mL)

6.0 *

600

0

IL-10

300

120 100 80 60

0.8 06 0.6 0.4 **

40 0.2

0.2 0

20 Control

1.0

5.0

mg/kg/day

0

Control

1.0

5.0

mg/kg/day

0

Control 1.0

5.0

mg/kg/day

Fig. 6 – Effect of LDH on cytokine production by spleen lymphocytes ex vivo. Spleen lymphocytes were prepared from mice administered with LDH at 5.0 mg/kg/day or 1.0 mg/kg/day for 14 days and incubated at 2.0 · 106 cells/mL in 5% FBS-RPMI 1640 medium containing ConA at 10 lg/mL for 48 h. The amounts of IL-4, IL-5, IL-6, IL-10, IFN-c, TNF-a, and TGF-b1 produced by spleen lymphocytes were measured by ELISA. Each group consisted of 5 mice. A bar represents the average of Ig levels of 5 mice. Statistically significant differences from control were represented as *p < 0.05 or **p < 0.01.

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cytokines promote the expansion of IgA class-switched B cells and their differentiation into IgA-secreting plasma cells (Chen & Cerutti, 2010; Lamm & Phillips-Quagliata, 2002; Mora & von Andrian, 2008). It was also confirmed that LDH did not affect Ig production by MLN lymphocytes in vivo (data not shown). Lymphocytes prepared from each tissue contained various sorts of lymphocytes. Although it is difficult to identify the sort of cells, which LDH works on first, it is definite that Ig-producing cells such as B cells and plasma cells were stimulated by LDH in certain ways. Probably the mechanisms that increase Ig production in vitro and in vivo are not the same. Increased Ig level in serum and in culture supernatant of both spleen lymphocytes and PP lymphocytes might be caused by IL-4, IL-5, and IL-10 that were enhanced by LDH, since IL-4, IL-5, and IL-10 are Th2 cytokines, which relate to stimulation of the humoral immunity such as Ig production. Because IFN-c is a Th1 cytokine, LDH might also stimulate the cellular immunity. TNF-a is produced by antigen-presenting cells such as macrophages and dendric cells and activates defense response against infection in short order. LDH might stimulate both the adaptive immunity conducted by B cells and T cells and the innate immunity by macrophages. On the other hand, TGF-b1 promotes differentiation of naive T cells to regulatory T cells, which relate to the convergence of immune response and the immune tolerance to food antigen. Regulatory T cells produce TGF-b1 and suppress differentiation and activation of Th1 cells and Th2 cells (Sakaguchi, 2010). It is possible that LDH might suppress TGF-b1 production or decrease the population of regulatory T cells and increase the amounts of Th1 and Th2 cytokines produced by lymphocytes. In conclusion, LDH increased IgA, IgG, and IgM production by spleen lymphocytes, MLN lymphocytes, and PP lymphocytes in vitro. From the results of oral administration with LDH to mice, LDH increased the IgA level in serum in the group administered at 5.0 mg/kg/day. In addition, IgA and IgG production by PP lymphocytes in the group administered at 1.0 mg/kg/day and IgA, IL-4, IL-5, IL-10, IFN-c, and TNF-a production by spleen lymphocytes from the group administered at 5.0 mg/kg/day was enhanced, indicating that LDH stimulated both Ig and cytokine production by lymphocytes in vivo. However, how LDH works to increase Ig production by mouse lymphocytes is still unclear. Therefore, further investigations are needed to make clear which kinds of lymphocytes LDH works on and how LDH interacts with various kinds of lymphocytes. LDH has been identified from tuna bulbus arteriosus as an active substance that exerts the immunomodulatory effect in the study for exploiting unutilized food resources (Daifuku et al., 2012). Thus, not only the fish flesh and meats but also unutilized parts of the fish and feeders such as viscera, these are all rich in LDH, would be exploitable for the practical use of LDH as functional foods that modulate the immune system, especially the intestinal immunity.

R E F E R E N C E S

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