Effects of early enteral nutrition supplemented with arginine on intestinal mucosal immunity in severely burned mice

Clinical Nutrition 29 (2010) 124–130

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Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu

Original Article

Effects of early enteral nutrition supplemented with arginine on intestinal mucosal immunity in severely burned mice Jun Fan a, Qingyan Meng a, *, Guanghua Guo b, Yong Xie c, Xuedong Li d, Yiping Xiu a, Tairan Li a, Liang Ma a a

Department of Burns, The Northern Hospital, 83 Wenhua Road, Shenyang 110015, Liaoning, China Burn Institute, The first affiliated hospital, Medical College of Nanchang University, 461 Bayi Road, Nanchang, 330006 Jiangxi, China c Digestion Institute, The first affiliated hospital, Medical College of Nanchang University, 461 Bayi Road, Nanchang, 330006 Jiangxi, China d Department of bone, The first affiliated hospital of Shantou University, 57 Changping Road, Shantou 515041, Guangdong, China b

a r t i c l e i n f o

s u m m a r y

Article history: Received 20 November 2008 Accepted 14 July 2009

Background: To investigate the effects of early enteral nutrition (EN) supplemented with Arginine (Arg) on intestinal mucosal immunity in severely burned mice. Methods: Forty-four mice were randomly assigned into four groups: a sham injury þ EN group (n ¼ 10), a sham injury þ EN þ Arg group (n ¼ 10), a burn þ EN group (n ¼ 12), and a burn þ EN þ Arg group (n ¼ 12) and the mice in two experimental groups received a 20% total body surface area (TBSA), fullthickness scald burn on the back. Then, the burned mice were given a 175 kcal/kg body wt/day of conventional enteral nutrition or an isonitrogenous and isocaloric enteral nutrition supplemented with Arg by gastric gavage for 7 days. There was isonitrogenous and isocaloric intake in two experimental groups. The mice in two control groups received the same procedures as above, except for burn injury. On day 7 after injury, all mice among four groups were euthanized and the entire intestine was harvested. Intestinal immunoglobulin A (IgA) levels, total lymphocyte yield, and lymphocyte subpopulations in Peyer’s patches were analyzed. Levels of IFN-g, IL-2, IL-4 and IL-10 in gut homogenates were also measured by ELISA. Results: Total lymphocyte yield, numbers of lymphocyte subpopulations, and intestinal IgA levels in the EN þ ARG group were higher than those in the EN group (p < 0.05). Levels of gut tissue cytokines were significantly altered with enteral Arg supplementation: levels of IL-4 and IL-10 were increased, and levels of IFN-g and IL-2 declined, when compared with the EN-fed mice (p < 0.05). Conclusions: The results of this study suggested that enteral nutrition supplemented with Arg has changed the cytokine concentrations in intestinal homogenates from a pro- to an anti-inflammatory profile, increased sIgA levels and changed lymphocytes in severely burned mice. Ó 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords: Arginine Enteral nutrition Peyer’s patches Cytokines Immunoglobulin A Burns

1. Introduction Arginine (Arg) has attracted great interest for its numerous useful physiologic properties and pharmacological role. Functionally, Arg stimulates the secretion of anabolic hormones such as growth hormone, prolactin, and insulin-like growth factor-1, which may enhance protein synthesis and wound healing.1 In addition, it is a major substrate for the production of nitric oxide (NO), which is toxic to tumors and infected cells 2,3 and is important precursor of polyamines (via Arginase) which are important for cellular division.2 Although Arg is synthesized endogenously from L-citrulline, under severe stress the systemic demand for Arg is markedly

* Corresponding author. Tel.: þ86 24 230 851291; fax: þ86 24 230 856448. E-mail address: [email protected] (Q. Meng).

increased, whereas its bioavailability is reduced. Therefore, Arg is classified as a semiessential amino acid and conditionally as an essential nutrient for severely injured patients or in stressed states.2 Numerous studies have demonstrated that Arg improves the immunocompetence. For example, dietary Arg increased the weight of the thymus in healthy animals.4 Intravenous infusion of Arg was also associated with an increase in the release of T cells from the thymus.5 In addition, Arg had a direct effect on T cell activity in vivo and in vitro.6,7 In one study, maximal proliferation of peripheral mononuclear blood cells occurred when the cells were cultured in medium containing Arg.8 Moreover, it had been observed that Arg supplementation could reduce gut mucosal injury,9 accelerate intestinal mucosal regeneration,10 enhance immunity,11–13 inhibit bacterial translocation,14 and improve survival15 in several animal models.

0261-5614/$ – see front matter Ó 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2009.07.005

J. Fan et al. / Clinical Nutrition 29 (2010) 124–130

The role of Arg supplementation in burns also continues to be explored. For instance, Yu YM et al. found that increased extrahepatic uptake of Arg contributed to accelerated urea production in burned patients further exacerbating its losses from the body.16 Stinnett et al17 reported that plasma Arg declined 30–40% after severe burn injury while dietary Arg supplementation increased the Arg level in plasma.16 In addition, Arg-supplemented diet could promote protein anabolism, attenuate muscle protein catabolism, decrease expression of inflammatory cytokines, and improve survival in burned rats.18,19 Besides, Arg supplementation attenuated oxidative stress and improved macrophage response after burn injury.20 However, until now, there are few studies that have addressed the importance of Arg on GALT function and intestinal IgA response after severe burn. We hypothesized that enteral Argsupplemented nutrition after burn injury might produce a different pattern of the function of Peyer’s patches and their ability to secrete immunoglobulin A (IgA) compared to a common enteral nutrition (EN). Since IgA-mediated intestinal immune response is controlled by the Th2 type IgA-stimulating and the Th1 type IgA-inhibiting cytokines, we also investigated the effects of enteral Arg supplementation on the Th2 type IgA-stimulating and the Th1 type IgA-inhibiting cytokines in burned mice. 2. Materials and methods 2.1. Animal preparation Male Balb/C mice weighing 20–25 g were used in this experiment. All mice were housed in stainless-steel cages maintained in a temperature and humidity-controlled room. Mice were allowed free access to a rodent chow and water for a 7-day acclimatization period. All procedures of this study were in accordance with the guide for the care and the use of laboratory animals published by the National Institute of Health and were approved by the Science and Technology Committee of our hospital. 2.2. Experimental burn injury and grouping Forty-four mice were randomly assigned into four groups: a sham injury þ EN group (n ¼ 10), a sham injury þ EN þ Arg group (n ¼ 10), a burn þ EN group (n ¼ 12), and a burn þ EN þ Arg group (n ¼ 12). The mice in two experimental groups received a fullthickness scald burn according to the method as previously described.21 Briefly, the mice were weighted and anesthetized by intraperitoneal (i.p.) injection of 60 mg/kg body weight pentobarbital sodium, and the entire dorsal surface was shaved and fixed in a mold with a hole in order to let 20% of their total body surface area (TBSA) be exposed on the shaved back. Then, the exposed area was immersed in 90  C water for 7 s to produce a full-thickness burn confirmed by histological evaluation. Immediately the burned mice received a 2-ml intraperitoneal (i.p.) injection of sterile 0.9% saline for fluid resuscitation with the application of an antibacterial agent (silver sulfadiazine cream, Hunan Erkang Pharmaceutical Co., Ltd, Table 1 Formulas of rodent diet. Component (units)

Rodent chow

Protein (g/100 g) Nitrogen (g/100 g) Fat (g/100 g) Carbohydrate (g/100 g) Water and fiber (g/100 g)

17 2.7 3 59 21

125

Table 2 Formulas of enteral nutrition. Component (units)

Enteral nutrition

Calorie(KJ) Casein (g/L) Nitrogen(g/L) Fat(g/L) Carbohydrate (g/L) Composition of total calorie Protein(kcal) Fat(kcal) Carbohydrate(kcal)

4184 38 5.7 2.8 210 150 30 820

Changsha, China) to the burned areas every 12 h. Since sensitive nervous terminations were destroyed by the full skin thickness burn, pain and discomfort associated with the burn wound are minimal. Analgesics after burn injury were not employed due to their undefined effects on immune function. The mice in two control groups received the same procedures as above, except for burn injury. 2.3. Nutrition support The enteral nutrition was administrated by gastric gavage at 2 h after injury and continued for 7 days. All mice among four groups received an identical enteral nutrition solution as their base formula, which provided, per liter, 1000 kcal, 38 g protein, 210 g carbohydrate, and 2.8 g fat. The supplied energy ratio of the enteral nutrition solution was 82% carbohydrate, 3% fat, and 15% protein and the proportion of calorie to nitrogen was 150:1. The mice in the sham þ EN þ Arg and burn þ EN þ Arg groups were then supplemented with 1 g/kg/d Arg. The dose of 1 g/kg/d Arg was chosen in accordance with previous study.22 To assure isonitrogenous intake among four groups, those in the sham þ EN and burn þ EN groups were supplemented with 1 g/kg/d tyrosine. All mice among four groups were given 175 kcal/kg body wt/day 23 and approximately 1 g N/kg body wt/day by gastric gavage. There was no difference in average calorie and nitrogen intake among four groups. A quarter of the daily requirement of energy was supplied within the first 24 h, and a half in the second 24 h. After the second day, full energy requirement was given. The enteral feedings were divided evenly 4 times per day and all mice were allowed to drink water freely (Tables 1–3). 2.4. Cell isolation On day 7 after injury, all mice were weighted and euthanized and a middle abdominal incision was made and the entire intestine was carefully removed. The number of Peyer’s patches from each small intestine removed was counted and lymphocytes were isolated from Peyer’s patches according to the method as previously described.24 In brief, Peyer’s patches were excised from the serosal side of the intestine and placed in Petri dishes containing

Table 3 Mice body weight and weight change (g). Group

n

Body weight before experiment (g)

Loss of body weight (g)

Sham þ EN Sham þ EN þ Arg Burn þ EN group Burn þ EN þ Arg group

10 10 12 12

23.5  3.1 22.8  2.6 23.7  3.5 24.1  2.3

0.3  0.1 0.2  0.1 3.5  1.4a 3.1  1.5a

Values are mean  SD. a versus Normal group, p < 0.05.

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J. Fan et al. / Clinical Nutrition 29 (2010) 124–130

2.5. Flow cytometric analysis

Table 4 PP number and total cell yield. Group

n

Number of Peyer’s patches

Total cell yield (  106)

Sham þ EN Sham þ EN þ Arg Burn þ EN group Burn þ EN þ Arg group

10 10 12 12

9.2  1.2 9.5  1.3 8.6  1.2 8.7  1.1

5.1  1.3 5.3  1.2 2.4  0.4a,b 3.2  0.6a

Values are mean  SD. a versus Normal group, p < 0.05. b versus EN þ ARG group, p < 0.05.

Table 5 Percentage of lymphocyte subpopulations (%). Group

n

CD3þ

CD4þ

CD8þ

Sham þ EN Sham þ EN þ Arg Burn þ EN group Burn þ EN þ Arg group

10 10 12 12

32.3  5.8 32.8  6.3 34.9  5.2 31.1  4.7

24.1  4.3 8.3  1.6 23.8  4.6 8.7  1.7 23.5  4.1 12.4  1.5a,b 22.4  3.8 8.5  1.8

2.6. Intestinal IgA levels

CD19þ 61.3  8.4 62.5  9.1 52.4  7.6a,b 59.7  8.2

Values are mean  SD. a versus Normal group, p < 0.05. b versus EN þ ARG group, p < 0.05.

The entire small intestine was flushed twice with a total of 10 ml of cold HBSS with 1%protease inhibitor (Sigma) and the intestinal washings were collected. After the intestinal washings were centrifuged at 800  g for 10 min, supernatants were harvested and stored at a 84  C freezer for IgA analysis. The intestinal washing IgA levels were assessed with a sandwich-type enzyme-linked immunosorbent assay, by using an a-chain specific goat anti-mouse IgA (Sigma) to coat the plate, a purified mouse IgA (Sigma) as standard, and an alkaline phosphatase conjugated anti-mouse IgA (Sigma) for detection, and were normalized for the length of each intestinal segment (IgA/cm intestine). 2.7. Cytokine assay From the proximal, middle, and distal small intestine, 0.6 g of intestines were removed and placed in 10 mL of lysis buffercontaining protease inhibitors (Sigma) at 4  C. Samples were homogenized for 30 s, and homogenates were ultracentrifuged at 15,000 rpm for 45 min at 4  C. The amount of cytokines (IFN-g, IL-2, IL-4, and IL-10) in the supernatants was measured by using an enzyme-linked immunosorbent assay. Capture and detection

loss of body weight (g)

A

25 20 15 10 5

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

7

C

12

6 5 4

a ab

3 2

B

a

Sham+ EN Sham+ EN+ Burn+ EN group Arg group group

Sham+ EN Sham+ EN+ Burn+ EN Burn+ EN+ Arg group group group Arg group

number of PPs

Total cell yield of PPs (x106)

body weight before experiment (g)

5 ml of cold Hank’s balanced salt solution (HBSS). The tissues were gently glass ground, and the cell suspension was passed through a 150 mesh stainless-steel screen into a beaker and transferred to a 15-ml conical centrifuge tube, centrifuged at 400  g for 10 min at room temperature. The pellet was disrupted and resuspended in 8 ml of HBSS, layered over 5 ml of 67% Percoll, and centrifuged at 600  g for 20 min at room temperature. The cells at the interface were harvested with Pasteur pipet and washed twice with HBSS, and resuspended in RPMI-1640 with antibiotics and fetal calf serum. Viability was assessed by trypan blue exclusion and total Peyer’s patch cell yield was determined with a hemocytometer. Cell counts were adjusted to 106/ml using HBSS and 1 ml of this cell suspension was placed into 1.5 ml microcentrifuge tubes for cell staining.

30

To detect the phenotypes of lymphocytes isolated from Peyer’s patches, 106 of cells were incubated with either isotypic control, or monoclonal antibodies (mAb) against CD3, CD4, CD8, or CD19 (Caltag Laboratories, Burlingame, CA) for 30 min on ice in the dark. All mAb were either conjugated with fluoresceinisothiocyanate (FITC) or phycoerythin (PE) or PE-Cy5 (TRI). After the incubated cells had been washed twice with cold HBSS, antigen expression was measured by flow cytometry (Becton-Dickinson Inc., San Jose, CA) and the total numbers of lymphocyte subpopulations in Peyer’s patches were calculated by multiplication of the cell ratio with total cell yield (Tables 4 and 5).25

a

Burn+ EN+ Arg group

D

10 8 6 4 2

1 0

0 Sham+ EN Sham+ EN+ Burn+ EN Burn+ EN+ Arg group Arg group group group

Sham+ EN Sham+ EN+ Burn+ EN Burn+ EN+ group Arg group group Arg group

Fig. 1. A,B,C and D represent the body weight before experiment, the loss of body weight, total Peyer’s patch cell yield, and number of Peyer’s patches on the small intestine in the sham injury þ EN group (n ¼ 10), sham injury þ EN þ Arg group (n ¼ 10), burn þ EN group (n ¼ 12), and burn þ EN þ Arg group (n ¼ 12), respectively. Different letters indicate a significant difference among groups: ap < 0.05 versus the two control groups, bp < 0.05 versus Burnþ EN þ Arg group. EN, enteral nutrition; Arg, arginine.

Number of lymphocyte subsets (x106)

J. Fan et al. / Clinical Nutrition 29 (2010) 124–130

127

Sham+ EN group Sham+ EN+ Arg group Burn+ EN group Burn+ EN+ Arg group

4 3.5 3

a

2.5 2

ab

a

1.5

ab

1

a

a

ab a

0.5 0 CD3+

CD4+

CD8+

CD19+

Fig. 2. numbers of PP lymphocyte subsets in the sham injury þ EN group (n ¼ 10), sham injury þ EN þ Arg group (n ¼ 10), burn þ EN group (n ¼ 12), and burn þ EN þ Arg group (n ¼ 12). Different letters indicate a significant difference among groups: ap < 0.05 versus the two control groups, bp < 0.05 versus Burnþ EN þ Arg group. EN, enteral nutrition; Arg, arginine.

anti-mouse IFN-g, IL-2, IL-4, and IL-10 Abs were purchased from Pharmingen (San Diego, CA). The concentration of each cytokine was calculated based on a standard curve obtained with the recombinant murine cytokine (Pharmingen). The amount of cytokines was determined from a standard curve and expressed as picograms per milliliter. 2.8. Statistical analysis Data were expressed as the mean  SD and differences among groups were analyzed by ANOVA using Newman-Keuls’ test. Values of P < 0.05 were regarded as significant. 3. Results 3.1. Body weight change As shown Fig. 1A, there were no differences in body weight at the beginning of the experiment among four groups. At the end of the experiment, the mice lost more body weight in the burn þ EN and burn þ EN þ Arg groups than in two control groups with no difference between both experimental groups (Fig. 1B.) 3.2. Peyer’s patches characterization and phenotyping

percentage of PP lymphocyte subsets (%)

As compared with two control groups, total Peyer’s patch cell yield in two experimental groups was significantly decreased (p < 0.05; Fig. 1C). However, the total Peyer’s patch cell yield was

higher in the burn þ EN þ Arg group than in the burn þ EN group (p < 0.05; Fig. 1C), while no difference was observed in the number of Peyer’s patches on the small intestine among four groups (P > 0.05; Fig. 1D). As shown in Fig. 2, burn injury resulted in an obvious reduction in B- and T-cell numbers (P < 0.05). But numbers of B and T cells were greater in the Burnþ EN þ Arg group than in the Burn þ EN group (P < 0.05). Also, a similar decrease in the number of CD4 and CD8 cells in Peyer’s patches was observed in burned mice (p < 0.05). Nevertheless, the number of CD4 and CD8 cells in Peyer’s patches markedly increased from the Arg-fed burned mice compared with the EN-fed burned mice (p < 0.05). Burn injury also led to changes in the percentage of lymphocyte subpopulations in Peyer’s patches: the percentage of CD19 cells was declined and the percentage of CD8 cells correspondingly increased, when compared with two control groups (p < 0.05; Fig. 3). Yet, Arg therapy restored the percentage of lymphocyte subpopulations in PPs to a sham control situation (Fig. 3).

3.3. Intestinal IgA levels To determine the impact of enteral Arg supplementation on IgA-mediated intestinal immunity in severely burned mice, the intestinal washing IgA levels were detected by ELISA. The result revealed that severe burn contributed to an obvious decrease in intestinal IgA levels, whereas enteral Arg supplementation blunted this change, and that IgA levels of intestinal washings were higher

Sham+ EN group Sham+ EN+ Arg group Burn+ EN group Burn+ EN+ Arg group

75 65 55

ab

45 35 25

ab

15 5 CD3+

CD4+

CD8+

CD19+

Fig. 3. The percentages of PP lymphocyte subsets in the sham injury þ EN group (n ¼ 10), sham injury þ EN þ Arg group (n ¼ 10), burn þ EN group (n ¼ 12), and burn þ EN þ Arg group (n ¼ 12). Different letters indicate a significant difference among groups: ap < 0.05 versus the two control groups, bp < 0.05 versus Burn þ EN þ Arg group. EN, enteral nutrition; Arg, arginine.

128

J. Fan et al. / Clinical Nutrition 29 (2010) 124–130

4. Discussion

70

IgA levels (ug/cm)

65 60

a

55 50

ab

45 40 35 30 25 20

Sham+ EN group

Sham+ EN+ Arg group

Burn+ EN group

Burn+ EN+ Arg group

Fig. 4. IgA levels in intestinal lavage fluid in the sham injury þ EN group (n ¼ 10), sham injury þ EN þ Arg group (n ¼ 10), burn þ EN group (n ¼ 12), and burn þ EN þ Arg group (n ¼ 12). Different letters indicate a significant difference among groups: ap < 0.05 versus the two control groups, bp < 0.05 versus Burnþ EN þ Arg group. EN, enteral nutrition; Arg, arginine.

in the burn þ EN þ Arg group than in the burn þ EN group (P < 0.05; Fig. 4).

3.4. Cytokine We also determined whether enteral Arg supplementation affected levels of IFN-g, IL-2, IL-4, and IL-10 in gut homogenates. As shown in Fig. 5, levels of gut tissue cytokines were significantly altered with Arg supplementation: levels of IL-4 and IL-10 in gut homogenates were increased whereas levels of IFN-g and IL-2 were declined, when compared with the EN-fed mice (p < 0.05).

A

B

ab

180

In this study, we observed that burn injury resulted in a significant decrease in total Peyer’s patch cell yield. Because Peyer’s patches (PPs) on the small intestinal wall are highly specialized lymphoid follicles with naive B cells, follicular dendritic cells (DCs), and T cell and act as the primary inductive sites for mucosal immunity, where naive lymphocytes are sensitized, migrate to mesenteric lymph nodes, then enter into system circulation via the thoracic duct, and ultimately home to various mucosal effector sites and perform the mucosal immune function,26,27 a significant decrease in total Peyer’s patch cell yield may impair the intestinal immune barrier, contributing to an increased translocation of bacteria and endotoxin from bowel lumen into systemic circulation. In current study, the total Peyer’s patch cell yield from the Arg-fed mice was significantly increased when compared with the EN-fed mice. Previous investigation also demonstrated that Arg supplementation could promote proliferation of total lymphocytes in Peyer’s patches and enhance immunoglobulin IgA secretion in septic rats.11 Thus, our findings indicated that an enteral nutrition supplemented with Arg could improve significantly total Peyer’s patch cell yield compared with a common enteral nutrition in burned mice. We also attempted to evaluate the effects of enteral Arg supplementation on the B cells (CD19), T cells (CD3), helper T cells (CD4), and cytotoxic T cells (CD8) in Peyer’s patches by flow cytometry. The results revealed that the numbers of all lymphocyte subsets in Peyer’s patches after injury were markedly reduced. Since B cells in Peyer’s patches are a major source of IgA precursor cells destined to repopulate in the lamina propria of the mucosa for production of IgA, the B-cell repertoire of Peyer’s patches forms the basis for IgA response in intestine.24 In addition, the development

160

a IL-2 (pg/ml)

IFN-r (pg/ml)

140 120 100 80 60 40 20

IL-4 (pg/ml)

C

200 180 160 140 120 100 80 60 40 20 0

Sham+ EN Sham+ EN+ Burn+ EN group Arg group group

Burn+ EN+ Arg group

D

ab

a

Sham+ EN group

Sham+ EN+ Arg group

Burn+ EN group

Burn+ EN+ Arg group

3000

a 2500

a ab

IL-10 (pg/ml)

0

50 45 40 35 30 25 20 15 10 5 0

ab

2000 1500 1000 500

Sham+ EN group

Sham+ EN+ Arg group

Burn+ EN group

Burn+ EN+ Arg group

0

Sham+ EN group

Sham+ EN+ Burn+ EN Arg group group

Burn+ EN+ Arg group

Fig. 5. A, B, C, and D represent levels of IFN-g, IL-2, IL-4, and IL-10 in gut homogenates in the sham injury þ EN group (n ¼ 10), sham injury þ EN þ Arg group (n ¼ 10), burn þ EN group (n ¼ 12), and burn þ EN þ Arg group (n ¼ 12), respectively. Different letters indicate a significant difference among groups: ap < 0.05 versus the two control groups, bp < 0.05 versus Burnþ EN þ Arg group. EN, enteral nutrition; Arg, arginine.

J. Fan et al. / Clinical Nutrition 29 (2010) 124–130 Table 6 T-cell and B-cell yields (106). Group

n

T cell

B cell

Sham þ EN Sham þ EN þ Arg Burn þ EN group Burn þ EN þ Arg group

10 10 12 12

1. 8  0.4 1. 7  0.3 0.8  0.1a,b 1.1  0.2a

3.2  0.7 3.3  0.5 1.3  0.2a,b 2.1  0.4a

Values are mean  SD. a versus Normal group, p < 0.05. b versus EN þ ARG group, p < 0.05.

Table 7 T-cell subsets (106). Group

n

CD4þ

CD8þ

Sham þ EN Sham þ EN þ Arg Burn þ EN group Burn þ EN þ Arg group

10 10 12 12

1.2  0.2 1.3  0.2 0.5  0.1a,b 0.8  0.1a

0.4  0.1 0.5  0.1 0.2  0.1a,b 0.3  0.1a

Values are mean  SD. a versus Normal group, p < 0.05. b versus EN þ ARG group, p < 0.05.

of PP B cells into IgAþ B cells appears to involve the class-switching to IgA, which is highly dependent on B-cell interaction with properly activated T cells.27 So, a marked reduction in the number of B and T cell populations in Peyer’s patches could negatively affect IgA-mediated intestinal mucosa response. Our results revealed that enteral Arg supplementation could attenuate these changes and the numbers of CD4, CD8, and CD19 cells in Peyer’s patches from the Arg-fed mice were significantly increased compared with the EN-fed mice. It is well known that IgA is the primary specific immunoglobulin that protects the intestinal mucosal surfaces. Intestinal IgA produced by the lamina propria plasma cells is transported by secretory component on the basement membranes of the mucosal cells onto the lumen, where it blinds or agglutinates pathogens and their toxins, and prevents their attachment to the mucosal surface. Without the attachment, infection does not occur. Therefore, intestinal IgA levels depend on normal numbers of functioning immunocompetent cells in the lamina propria mucosa and a cytokine milieu appropriate to the production of IgA.27 In fact, these functioning immunocompetent cells in the lamina propria mucosa mainly derived from Peyer’s patches,24 which supports the statement that Peyer’s patches play a central role in coordinating and maintaining the intestinal IgA response.27 In this study, we confirmed that severe burn resulted in an obvious reduction in intestinal IgA levels, while Arginine therapy could markedly increase intestinal IgA levels, probably owing to augmented B and T cell counts (Tables 6–9). IgA-mediated intestinal immune response is controlled by the Th2 type IgA-stimulating and the Th1 type IgA-inhibiting cytokines. Th1 cytokines such as IL-2 and IFN-g, which enhance cell-mediated immunity, are produced by Th1 lymphocytes.

129

A predominant Th1 effect results in activation of T lymphocytes. Th2 cytokines such as IL-4 and IL-10, which enhance humoral immunity, are produced by Th2 lymphocytes. A predominant Th2 effect results in activation of B lymphocytes and up-regulation of antibody production. The effects of Th1 or Th2 lymphocytes are counter-regulatory.11,27 It appears that this balance between the Th2 and the Th1 type cytokines plays a critical role in IgA control.27 To clarify the influence of Arg treatment on local gut cytokine secretions, levels of IL-2, IFN-g, IL-4, and IL-10 in gut homogenates were analyzed by ELISA. The results disclosed that there was a shift from Th1 to Th2 profile of cytokine in gut homogenates from the Arg-fed mice: levels of IL-4 and IL-10 were significantly increased and levels of IL-2 and IFN-g declined, when compared to the EN-fed mice. Because IL-4 stimulates B cells to switch to IgAþ cells within the Peyer’s patches and IL-10 promotes conversion of sIgAþ B cells to mature sIgA-secreting plasma cells in the lamina propria,27 Th2 type cytokines may play an important role in stimulating intestinal IgA secretion. Our results also revealed that both IL-4 and IL-10 levels correlated excellently with intestinal IgA levels. Cui et al.19 confirmed that an Arg- supplemented diet decreased tissue inflammatory cytokine expression and improved survival in burned rats. Shang HF et al11 reported that Arg-supplemented diet increased splenocytes IL-10 production in vitro. As to possible mechanisms by which Arg induces a Th2-like cytokine response, there is accumulating evidence indicating that NO seems to modulate phenotype of T helper cells and to promote a shift from Th1 to Th2, accompanied by a decline in proinflammatory mediators including IFN-g and IL-2 and by an increase in anti-inflammatory mediators such as IL-4 and IL-10.28–32 Indeed, Arg is the sole precursor of NO in most mammalian cells. It is a simple and unstable free radical, and has been identified as a potent intracellular mediator.20 Cui et al19 found that NO production increased in the supernatant of cultured splenic lymphocytes in the Arg group. Since we did not measure NO levels in gut, which was probably a shortcoming of this study, whether Arg-derived NO plays a role in modulating cytokine production requires further investigation. We recognize that the lack of functional data (gut permeability, responsiveness to any other challenge) should be acknowledged as limitations in this study. Because in the present study we focused on the influence of early enteral Arg supplementation on PP lymphocyte subsets, intestinal IgA levels and local inflammatory response in severely burned mice, influences of enteral Arg supplementation on functional data including gut permeability would be further assessed in future study. With respect to the dose of Arg, there are different reports from other study groups. In present study, the dose of 1 g/kg/d Arg was chosen in accordance with previous study.28 In that study, it was documented that excessive dosage (>2.4 g/kg body wt/d Arg) led to an increased morality, that low dose (<0.4 g/kg body wt/d Arg) was ineffective, and that 1.2 g/kg body wt/day Arg was appropriate, which was confirmed by our preliminary experiment as well.

Table 8 Cytokines (pg/ml). Group

n

IFN-g

IL-2

IL-4

IL-10

Sham þ EN Sham þ EN þ Arg Burn þ EN group Burn þ EN þ Arg group

10 10 12 12

51.2  15.4 48.6  16.2 135.8  35.1a,b 91.5  24.5a

17.6  4.3 15.4  5.1 38.5  8.2a,b 21.4  6.3a

149.5  36.2 135.4  33.5 57.2  18.9a,b 93.6  32.4a

2405.9  428.4 2384.3  401.6 1479.4  318.9a,b 1815.7  353.6a

Values are mean  SD. a versus Normal group, p < 0.05. b versus EN group, p < 0.05.

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J. Fan et al. / Clinical Nutrition 29 (2010) 124–130

Table 9 Intestinal IgA levels (ug/ml). Group

n

IgA levels

Sham þ EN Sham þ EN þ Arg Burn þ EN group Burn þ EN þ Arg group

10 10 12 12

53.8  9.2 56.1  8.6 34.7  7.2a,b 42.5  8.1a

Values are mean  SD. a versus Normal group, p < 0.05. b versus EN þ ARG group, p < 0.05.

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