Effects of swine gut antimicrobial peptides on the intestinal mucosal immunity in specific-pathogen-free chickens

Effects of swine gut antimicrobial peptides on the intestinal mucosal immunity in specific-pathogen-free chickens D. Wang,*† W. Ma,‡ R. She,*1 Qu. Sun,* Y. Liu,* Y. Hu,* L. Liu,* Y. Yang,* and K. Peng§ *College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; †Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, 200032, China; ‡College of Animal Science and Technology, Shandong Agricultural University, Taian, 271018, China; and §College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China pared with the control (P < 0.05). (3) Compared with the control, goblet cells were increased significantly in duodenum and jejunum from d 35 to 49 (P < 0.05) after SGAMP treatment and in the ileum were increased from d 21 to 49 (P < 0.05). (4) Swine gut antimicrobial peptides upgrade the expression of secretory IgA at different sites within the intestinal tract. The results strongly support that SGAMP can enhance the intestinal mucosal immune parameters of specific-pathogenfree chickens. Our research contributes to the further understanding of immunoregulatory mechanisms of intestinal mucosal immunity and the contribution of SGAMP to this process.

Key words: antimicrobial peptide, mast cell, intraepithelial lymphocyte, goblet cell, secretory immunoglobulin A 2009 Poultry Science 88:967–974 doi:10.3382/ps.2008-00533

INTRODUCTION

cock and Scott, 2000; Oppenheim et al., 2003). At present, many different peptides have been discovered in plants, insects, and animals (http://www.bbcm.units. it/~tossi/). Importantly, many antimicrobial peptides have been isolated from neutrophil, white cells, blood, tongue, skin, and intestine of swine (Agerberth et al., 1991; Selsted et al., 1992; Heller et al., 1998). Antimicrobial peptides include defensins, cathelicidin, histatins, cathepsin G, azurocidin, chymase, eosinophil-derived neurotoxin, lactoferrin, and many others. Many studies have concentrated on the role of antimicrobial peptides in innate immunity, such as killing a broad spectrum of microbes including gram-negative and gram-positive bacilli, fungi, and selected enveloped viruses (Boman, 1995; Oppenheim et al., 2003). Human neutrophil peptide defensins can promote acquired systemic immune response (Lehrer and Ganz, 1999) through T-cell-dependent cellular immunity and antigen-specific Ig production (Tani et al., 2000; Brogden et al., 2003). Additionally, some mammalian antimicrobial peptides have been shown to have a second major function of acting as a bridge between the innate immune system and the acquired system (Agerberth et

Multicellular organisms express numerous antimicrobial peptides, which are small peptides with fewer than 100 amino acid residues, and ubiquitously distributes at biological boundaries to aid host defenses. These polypeptides, originally described in silk worms (Steiner et al., 1981), now have been discovered extensively in all species, from plant seeds and insects to animals (Selsted and Ouellette, 1995; Lehrer and Ganz, 1999; Schröder and Harder, 1999; Ganz, 2003; Yeaman and Yount, 2003; MacDonald and Monteleone, 2005). Antimicrobial peptides are produced by specialized metabolic pathways in nature, and antimicrobial peptide are encoded by genes and produced by many cells, such as neutrophils, leucocytes, and epithelial cells lining the environmental interface of the gastrointestinal tract, urogenital tract, tracheobronchial tree, and skin (Han©2009 Poultry Science Association Inc. Received December 4, 2008. Accepted January 8, 2009. 1 Corresponding author: [email protected] or wdc268@yahoo. com.cn

967

Downloaded from http://ps.oxfordjournals.org/ at Serials Department on October 15, 2014

ABSTRACT Sixty specific-pathogen-free chickens were randomly divided into 2 groups (30 chickens for each group) to determine the effect of swine gut antimicrobial peptides (SGAMP) on intestinal mucosal immunity. All chickens were raised in negative-pressure isolators and fed the same diet. The results were as follows. (1) In the SGAMP group, the number of mast cells was increased markedly in the duodenum from d 21 to 49 (P < 0.05), and similar results were observed in the jejunum and ileum after being treated with SGAMP (P < 0.05). However, fewer mast cells were observed in those same tissues in the control. (2) Intraepithelial lymphocytes in the duodenum, jejunum, and ileum were increased significantly from d 21 to 49 in the SGAMP group com-

968

Wang et al.

MATERIALS AND METHODS Isolation of Swine Intestinal Antimicrobial Peptides Swine gut antimicrobial peptides were isolated from swine intestine by acetic acid extraction (Ghosh et al., 2002; Ma et al., 2004). Swine intestines were washed with sterile saline (0.9% sodium chloride). The chorion and fat were removed and mucosa were isolated from the intestine. The mucosa was homogenized by a muller in ice-cold aqueous 5% acetic acid (1:10 wt/vol) that contained 1 mmol/L of protease inhibitor phenylmethyl sulfonyl fluoride (Amresco, Solon, OH). The extracts were placed in a boiling water bath for 20 min, then after rapid cooling, the precipitate was discarded by centrifugation at 6,440 × g for 30 min at 4°C. The clarified extracts were sonicated for 30 s on ice and stirred overnight at 4°C. Ice-cold 5% acetic acid (1:1 vol/vol) was added in the presence of phenylmethyl sulfonyl fluoride and extraction was made overnight at 4°C. The extract was centrifuged at 6,440 × g for 30 min at 4°C and the pH of clarified extract was adjusted to 6.0 with sodium hydroxide. The precipitates that formed were removed by centrifugation (6,440 × g for 30 min at 4°C). Then the clarified extract was loaded on a 10 × 300 mm Sephadex G-100 column and G-25 column (GE Life Science Co., Uppsala, Sweden) and eluted by 0.2 mol/L of sodium acetate buffer. The SGAMP elution was analyzed by agarose diffusion assay (Lehrer et al., 1991) and Pasteurella cuniculicida was used as the test organism. Following, we identified the relative activity of this peptide under different temperature and pH ranges. The fractions of interest were purified with tricine-PAGE (Schagger and von Jagow, 1987) and sub-

jected to study its features. Fractions of interest were collected and freeze-dried before storage at 0°C for future use.

Birds and Experimental Treatments Sixty 1-day-old SPF chickens (Lohmann Brown, Merial Co. Ltd, Beijing, China) were used in this experiment. These chickens were negative for Newcastle disease virus and infectious bursal disease virus. They were assigned to 1 of 2 groups: 30 chickens in the SGAMP group and the others as the control group. Each chicken in the SGAMP group was inoculated with 0.1 mL (100 ug/mL) of SGAMP dilution by means of injection into the musculi colli on d 7, 14, 21, 28, 35, and 42. In the control group, chickens received the same volume of sterilize saline solution. Each group was separated in individual negative-pressure isolators. All chickens were provided feed and water ad libitum. This study was approved by the Institutional Animal Care and Use Committee of China Agricultural University.

Sampling Five chickens from each group were randomly sampled on d 7, 21, 35, and 49. Duodenum, jejunum, and ileum were collected and fixed in 2.5% (vol/vol) glutaraldehyde-polyoxymethylene solution immediately after chickens were weighed and killed.

Immunological Assays Measurement of the Contents of Mast Cells and Intraepithelial Lymphocytes. Intestines were fixed in 2.5% (vol/vol) glutaraldehyde-polyoxymethylene for 1 wk and then dehydrated and embedded in paraffin by routine methods. The serial paraffin sections (5 μm) were prepared with the laboratory protocol. Some sections were processed with common hematoxylin-eosin staining for intraepithelial lymphocyte (IEL) identification; the others were assayed for mast cells by staining with an improved toluidine blue staining route based on previous reports (Carlson and Hacking, 1972; Xu et al., 2001; Wang et al., 2008). The sections were incubated in 3 consecutive washings in xylol for 5 min to remove paraffin and then hydrated with 5 consecutive alcohol treatments: 100, 100, 95, 80, and 70%, respectively. The sections were immediately washed with distilled water for 1 min, and the sections were immersed in 0.8% toluidine blue (Sigma Co., Beijing, China) for 15 s. Slides were rinsed in distilled water and put into 95% alcohol until the mast cells appeared deep reddish purple under the microscope. The sections were immersed in alcohol: 100%, 100%, alcohol-xylol (1:1 vol/ vol), xylol, and xylol for 3 min, respectively and were then mounted with neutral gums. Five fields of view were selected to count the number of IEL and mast cells in different intestine under an Olympus light microscope (Melville, NY) and the mean

Downloaded from http://ps.oxfordjournals.org/ at Serials Department on October 15, 2014

al., 1991; Chertov et al., 1996; Chan and Gallo, 1998; Korthuis et al., 1999). Their presence on epithelial surfaces provides a powerful early response to microbial infection. Research has demonstrated that antimicrobial peptides are multifunctional effectors whose actions extend beyond inhibiting microbial proliferation. These functions include regulation of cell proliferation, extracellular matrix production, and cellular immune responses (Agerberth et al., 1991; Shi et al., 1994). Recently, we demonstrated that the oral administration of rabbit sacculus rotundus antimicrobial peptides could considerably modify the structure of the intestine and mucosal immunity immune parameters in healthy chickens (Liu et al., 2008). Therefore, the present study was conducted to evaluate the regulatory effect of swine gut antimicrobial peptides (SGAMP) on intestinal mucosal immunity in specific-pathogenfree (SPF) chickens. Moreover, the regulatory effect of SGAMP on intestinal immune cells was explored to better understand the innate immune mechanisms in the gut. Moreover, this study describes the efficacy of the use of antimicrobial peptides in protecting animal health and modulating animal immunity.

SWINE ANTIMICROBIAL PEPTIDES AND INTESTINAL MUCOSAL IMMUNITY

scope. The results were expressed as the number of cells per square millimeter.

Statistical Analysis Experimental data were analyzed by SPSS 13.0 (SPSS Inc., Chicago, IL) statistical program. The results were expressed as means and SE. Differences were considered significant at P < 0.05 or P < 0.01.

RESULTS Isolation and Characterization of SGAMP The SGAMP was extracted by acetic acid and purified by Sephadex G-100 gelatin, Sephadex G-25 gelatin, and tricine SDS-PAGE. The second peak in Sephadex G-100 gelatin and Sephadex G-25 gelatin had antibacterial activity after purification (Figure 1). The tricine SDS-PAGE demonstated that the molecular weight of this SGAMP was 5,972 Da. Swine gut antimicrobial peptide appears as a propiece and mature peptide when extracted from intestine (Figure 1). The nature of the SGAMP was characterized under different conditions of pH values and temperatures. It appears that activity of SGAMP was stable from 40 to 90°C and only decreased at 100°C. The antibacterial activity of SGAMP was pH-dependent with a bell curve-type response to pH. Peak antibacterial activity of SGAMP was seen at pH 7 (Figure 2).

Effect of SGAMP on Growth Performance of Chickens There were significant differences between the control and SGAMP group on growth performance in chickens (Table 1). Average daily weight gain and average weight gain in the first through the sixth week were significantly higher in the SGAMP group compared with the control group. Although ADFI did not differ between these 2 groups, G:F was significantly improved in the SGAMP group compared with the control group.

Effect of SGAMP on the IEL, Mast Cells, and Goblet Cells in Different Intestine Mast cells presented typical features of deep reddish purple under the microscope (Figure 3). They were mainly distributed in the mucosal lamina propria of duodenum, jujenum, and ileum. Compared with the control, the mast cell population was increased markedly in duodenum from d 21 to 49 after SGAMP administration (P < 0.05). Simultaneously, the mast cell increased in the jejunum and ileum at the age of 35 to 49 d (P < 0.05; Table 2). The IEL were unevenly distributed among endothelial cells of the intestinal epithelium (Figure 3). The IEL in duodenum, jejunum, and ileum were increased on d

Downloaded from http://ps.oxfordjournals.org/ at Serials Department on October 15, 2014

was calculated. The whole section was scanned for general qualitative observations, but detailed examination focused on mast cells and IEL. Measurement of Goblet Cells in Intestine with Periodic Acid-Schiff Staining. Goblet cells are the major source of mucin and are usually displayed with periodic acid-Schiff staining (Shatos et al., 2003). The serial histological sections (5 μm) were prepared using the same protocol described previously. After dewaxing and immediately washing with distilled water for 1 min, the specimens were immersed in 0.5% periodate solution (Sigma Co.) for 5 min at room temperature in the dark. Afterward, sections were immediately washed (30 s × 2) and soaked in Schiff’s solution at 37°C. After 60 min, sections were washed twice with a sufuric acid solution then quickly rinsed with distilled water. The subsequent steps followed the routine protocols of the laboratory. The number of goblet cells at different intestine sections was counted under an Olympus light microscope. Glycogen appears as a prune color and glucoprotein as pink. Expression of Secretory IgA with Immunohistochemistry Staining. Expression of secretory IgA (sIgA) in the tissue samples was performed by immunohistochemical analysis. The histological sections (5 μm) were prepared using the same protocol described above. First, the sections were heated at 120°C for 10 min, naturally cooled for 30 min, then incubated with 3% aqueous hydrogen peroxide for endogenous peroxidase ablation at room temperature for 30 min. Afterward, the following steps were executed in a moist chamber. The sections were incubated with blocking buffer containing 20% normal donor bovine serum and 80% PBS (0.01 M, pH 7.4) at 37°C for 30 min. The bovine serum was discarded, the sections were incubated with the serum of mouse anti-chicken IgA monoclonal antibody (Southern Biotechnology Inc., Birmingham, AL) for 2 h at 37°C, and the monoclonal antibody was used at a dilution of 1:100 in PBS (0.01 M PBS, pH 7.4), which gave optimal staining. After 2 h at 37°C, the sections were washed in PBS (3 × 5 min) and subsequently incubated for 1 h at 37°C with biotinylated goat antimouse IgG (Sigma Co.). After washing in PBS (3 × 5 min), the sections were incubated with Extr-Avidin conjugated horseradish peroxidase (Beijing Zhong Shan Golden Bridge Biotechnology Co. Ltd., Beijing, China) for 30 min at 37°C. Then the sections were incubated with 3,3-diaminobenzidine (Zymed Co.) for 12 min and kept at room temperature after being washed in PBS (3 × 5 min). After rinsing (3 × 5 min) in PBS, the tissues were counterstained with hematoxylin and dehydrated, cleared, and mounted with neutral gums. In parallel, tissue specimens in which the primary antibodies were omitted served as negative controls. Specificity was established by demonstrating the loss of immunoreactivity in matched tissue sections after competition. The number of positive cells in the intestinal lamina propria was counted under an Olympus light micro-

969

970

Wang et al.

21 to 49 in SGAMP group compared with the control (P < 0.05). In both the control and SGAMP groups there were, in general, more IEL found in the duodenum compared with the jejunum and ileum (Table 2).

Goblet cells were mainly distributed among columnar cells and presented a typical goblet shape (Figure 3). The quantity of goblet cells in duodenum and jejunum increased markedly from d 35 to 49 in the SGAMP-

Table 1. Average weight gain weekly and daily and G:F ratio (mean± SE, n = 10)1 Index Average weight gain in first week (g) Average weight gain in second week (g) Average weight gain in third week (g) Average weight gain in fourth week (g) Average weight gain in fifth week (g) Average weight gain in sixth week (g) Average weight gain daily (g) Average feed consumption daily (g) G:F ratio

SGAMP-treated group 70.35 83.63 82.59 85.30 119.13 117.21 15.54 29.82 2.27

± ± ± ± ± ± ± ± ±

2.06** 4.35** 2.14** 3.44** 5.40* 4.30* 0.20* 1.05 0.04*

Control group 51.80 61.75 53.89 59.21 101.43 110.71 11.10 28.85 2.60

± ± ± ± ± ± ± ± ±

2.54 3.35 2.57 2.44 4.71 5.68 0.19 0.79 0.05

1 Performance of average weight daily and G:F ratio between the swine gut antimicrobial peptide (SGAMP) and control group. T = SGAMP group; C = control group. * and **Significant difference from the control at P < 0.05 and P < 0.01, respectively.

Downloaded from http://ps.oxfordjournals.org/ at Serials Department on October 15, 2014

Figure 1. The purity determination of swine gut antimicrobial peptides (SGAMP) by using Sephadex G-100 gelatin, Sephadex G-25 gelatin, and tricine SDS-PAGE. A: The preparation of SGAMP by different routines. B: The purity determination of antibacterial peptides by using Sephadex G-100 gelatin and Sephadex G-25 gelatin. C: I = standard molecular weight marker; II = the ultrafiltrated pepetides; III = peptides chromatographed on Sephadex G-25; IV = peptides chromatographed on Sephadex G-100; V = the crude extract.

971

SWINE ANTIMICROBIAL PEPTIDES AND INTESTINAL MUCOSAL IMMUNITY

treated group (P < 0.05). Simultaneously, the number of goblet cells increased in ileum from d 21 to 49 (P < 0.05; Table 2).

Effect of SGAMP on the Contents of sIgA in Duodenum, Jujenum, and Ileum The positive signals of sIgA mainly distributed in the cytoplasm. The sIgA-positive cells were mainly distributed in the area of mucosal lamina propria of the duodenum (Figure 4). Although the effects of SGAMP on sIgA were variable in different intestinal sections, there was a general tendency that SGAMP could improve the level of sIgA at different intestinal sections during the whole experimental period compared with the control (P < 0.05 or P < 0.01; Figure 5).

Biological organisms have developed many strategies to compete against microbial pathogens. One of the most rudimentary, innate defense mechanisms is the cellular production of natural antimicrobial peptides (Selsted et al., 1984, 1993; Selsted and Harwig, 1987; Lehrer and Ganz, 1990; Couto et al., 1992; Gabay, 1994). A large body of evidence indicates that these peptides play a major role in pathogen protection. These peptides are produced by a diverse array of cell types including cells of the immune system (Selsted et al., 1984), small intestine (Lee et al., 1989; Agerberth et al., 1991, 1993), and skin of amphibians (Zasloff, 1987; Simmaco et al., 1994). The intestinal tract is the crossroad between the needs of nutrient absorption and host defense (MacDonald and Monteleone, 2005). It has been suggested that the gut has the most important role in the maintenance of homeostasis of the body (Guy-Grand et al., 1984; MacDonald and Monteleone, 2005). As a complicated immune system tissue, the intestinal tract plays a critical role in the first line of defense against ingested patho-

Figure 2. Effect of temperature and pH value on the antibacterial activity of swine gut antimicrobial peptides (SGAMP). A: The temperature range from 40 to 100°C on the antibacterial activity of SGAMP; we found that the SGAMP activity had slight difference. B: The pH value range from 4 to 10 had different influence on the SGAMP activity, its activity reached the peak at pH 7.

gens. The main site of the mucosal immune system in the intestine is referred to as gut-associated lymphoid tissue, and immunoassociated cells, including mast cells, goblet cells, and IEL, are involved in many processes to prevent pathogen invasion. The collaboration of those immunocompetent cells and natural antimicrobial peptides help the animals to compete against all kinds of infectious pathogens (Nizet et al., 2001; MacDonald and Monteleone, 2005). To our knowledge, little work

Table 2. Contents of intraepithelial lymphocytes (IEL), mast cells, and goblet cells in different intestines1 IEL (per 100 epithelial cells) Day

Intestine

7

Duodenum Jejunum Ileum Duodenum Jejunum Ileum Duodenum Jejunum Ileum Duodenum Jejunum Ileum

21 35 49

T 8.9 6.8 8.9 18.6 15.4 15.8 25.4 21.7 18.3 34.7 23.5 24.4

± ± ± ± ± ± ± ± ± ± ± ±

2.6 1.8 2.5 4.2* 2.2* 3.7* 3.5** 2.6** 3.2* 4.8** 2.8** 3.8**

C 8.6 7.0 9.2 14.2 10.5 12.4 17.5 12.6 14.5 25.4 14.2 16.5

± ± ± ± ± ± ± ± ± ± ± ±

T 2.3 1.5 2.1 3.4 1.9 3.6 3.8 2.2 3.9 4.2 2.5 4.0

Goblet cell (per 100 epithelial cells)

Mast cells

162.4 81.9 67.3 782.6 110.6 108.2 1,149.3 304.8 286.5 1,687.9 372.3 356.9

± ± ± ± ± ± ± ± ± ± ± ±

C 13.5 8.2 8.5 22.8* 10.8 10.3 28.9* 15.4* 12.5** 30.8** 18.9* 14.8**

145.0 82.4 68.6 674.6 105.2 95.3 1,056.7 265.8 221.8 1,534.2 354.2 302.4

± ± ± ± ± ± ± ± ± ± ± ±

T 12.4 8.5 6.2 15.4 11.3 8.5 18.3 16.1 13.8 26.8 17.5 15.2

14.3 16.2 19.0 19.3 20.5 24.8 24.8 29.2 33.5 31.5 34.6 39.4

± ± ± ± ± ± ± ± ± ± ± ±

2.2 2.1 2.5 2.6 2.5 2.9* 3.4* 3.0* 3.1** 3.5* 2.8* 3.5**

C 13.2 16.4 18.5 17.8 18.9 22.4 20.4 24.5 27.9 26.5 29.3 32.2

± ± ± ± ± ± ± ± ± ± ± ±

2.4 2.4 2.6 2.8 2.3 2.3 2.6 3.2 3.8 3.8 3.4 3.6

1 Contents of IEL, mast cells, and goblet cells in duodenum, jejunum, and ileum were examined on d 7, 21, 35, and 49. T = swine gut antimicrobial peptide group; C = control group. * and **Significant difference from the control at P < 0.05 and P < 0.01, respectively.

Downloaded from http://ps.oxfordjournals.org/ at Serials Department on October 15, 2014

DISCUSSION

972

Wang et al.

has been conducted on antimicrobial peptides from the swine gut on intestinal mucosal immunity. Thus, the present study aimed to determine the effect of SGAMP on the intestinal immunocompetent cells and mucosal sIgA-producing cells of SPF chickens. Our observations indicate that the concentrations of SGAMP used in this study are not toxic for chickens (Table 1). Further, our data showed that SGAMP could effectively enhance the contents of IEL, mast cells, and goblet cells in the intestine. It is well known that IEL, mast cells, and goblet cells are the major intestinal immunocompetent cells, which provide the first interface between the external and the internal microenvironments of the gastrointestinal tract. Previous reports

demonstrated that IEL were distributed extensively in the mucosa of different species, suggesting that IEL mediate the mucosal immune response and participate in the mucosal immune defense (Cerf-Bensussan et al., 1985; Duncker et al., 2006). Intraepithelial lymphocytes can offer effective protection against bacteria and viruses through the secretion of cytokines (Yang et al., 2003). Mast cells are important immunocompetent cells in the intestinal mucosal immune response that exert multifunctional roles by releasing prestored and de novo synthesized mediators such as histamine, proteases, serotonin, and others (Metcalfe et al., 1997). Some studies indicate that intestinal mucosal mast cells play an important role in the local mucosal immune re-

Figure 4. Distribution of secretory IgA (sIgA) positives in duodenum. Distribution of sIgA-positive signals between the control and swine gut antimicrobial peptide (SGAMP)-treated intestine. A: Distribution of duodenum sIgA-positive signals in the control group. B: Distribution of duodenum sIgA-positive signals in the SGAMP-treated chickens (immunohistochemistry staining, ×40).

Downloaded from http://ps.oxfordjournals.org/ at Serials Department on October 15, 2014

Figure 3. Distribution of mast cells, intraepithelial lymphocytes (IEL), and goblet cells in intestine. Number of mast cell, IEL, and goblet cells in the control (A to C) and the swine gut antimicrobial peptides (SGAMP)-treated group (D to F). A: Few mast cells present reddish purple and are distributed in mucosal lamina propria of duodenum (toluidine blue staining, ×20). B: Intraepithelial lymphocytes are distributed among the columnar cells [hematoxylin and eosin staining (HE), ×40]. C: Goblet cells present typical goblet shape and are distributed among columnar cells [periodic acid-Schiff (PAS) staining, ×20]. D: Many mast cells are distributed in the lamina propria of duodenum in the SGAMP group (toluidine blue staining, ×20). E: Intraepithelial lymphocytes in the SGAMP group (HE staining, ×40). F: Increased number of goblet cells in the SGAMP group (PAS staining, ×20).

SWINE ANTIMICROBIAL PEPTIDES AND INTESTINAL MUCOSAL IMMUNITY

973

Figure 5. Effect of swine gut antimicrobial peptide (SGAMP) on the secretory IgA (sIgA) level of duodenum, jejunum, and ileum (mean ± SE, n = 5) Content of sIgA positives in duodenum, jejunum, and ileum on d 7, 21, 35, and 49. The * and ** indicate significant difference from the control at P < 0.05 and P < 0.01, respectively. C = control group; T = SGAMP-treated group.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant. 30471301).

REFERENCES Agerberth, B., A. Boman, M. Andersson, H. Jornvall, V. Mutt, and H. G. Boman. 1993. Isolation of three antibacterial peptides from pig intestine: Gastric inhibitory polypeptide (7-42), diazepambinding inhibitor (32-86) and a novel factor, peptide 3910. Eur. J. Biochem. 216:623–629. Agerberth, B., J. Lee, T. Bergman, M. Carlquist, H. G. Boman, V. Mutt, and H. Jornvall. 1991. Amino acid sequence of PR~39: Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. Eur. J. Biochem. 202:849–854. Boman, H. G. 1995. Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13:61–92. Brogden, K. A., M. Heidari, R. E. Sacco, D. Palmquist, J. M. Guthmiller, G. K. Johnson, H. P. Jia, B. F. Tack, and J. P. B. McCray. 2003. Defensin-induced adaptive immunity in mice and its potential in preventing periodontal disease. Oral Microbiol. Immunol. 18:95–99. Caldwell, D. J., H. D. Danforth, B. C. Morris, K. A. Ameiss, and A. P. McElroy. 2004. Participation of the intestinal epithelium and mast cells in local mucosal immune responses in commercial poultry. Poult. Sci. 83:591–599. Carlson, H. C., and M. A. Hacking. 1972. Distribution of mast cells in chicken, turkey, pheasant, and quail, and their differentiation from basophils. Avian Dis. 16:574–577. Cerf-Bensussan, N., D. Guy-Grand, and C. Griscelli. 1985. Intraepithelial lymphocytes of human gut: Isolation, characterisation and study of natural killer activity. Gut 26:81–88. Chan, Y. R., and R. L. Gallo. 1998. PR-39, a syndecan-inducing antimicrobial peptide, binds and affects p130cas. J. Biol. Chem. 273:28978–28985. Chertov, O., D. F. Michiel, L. Xu, J. M. Wang, K. Tani, W. J. Murphy, D. L. Longo, D. D. Taub, and J. J. Oppenheim. 1996. Identification of defensin-1, defensin-2, and CAP37 1azurocidin as T-cell chemoattractant proteins released from interleukin-8stimulated neutrophils. J. Biol. Chem. 271:2935–2940. Couto, M. A., S. S. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer. 1992. eNAP-2, a novel cysteine rich bactericidal peptide from equine leukocytes. Infect. Immun. 60:5042–5047. Deplancke, B., and H. R. Gaskins. 2001. Microbial modulation of innate defense: Goblet cells and the intestinal mucus layer. Am. J. Clin. Nutr. 73:1131S–1141S. Duncker, S. C., A. Lorentz, B. Schroeder, G. Breves, and S. C. Bischoff. 2006. Effect of orally administered probiotic E. coli strain

Downloaded from http://ps.oxfordjournals.org/ at Serials Department on October 15, 2014

sponse (Caldwell et al., 2004; Morris et al., 2004). Goblet cells distributed throughout the intestine (Specian and Oliver, 1991) are capable of the maintenance of the protective mucus blanket by synthesizing and releasing mucins. The mucus constitutes the innermost layer and functions as a dynamic defensive barrier in response to intestinal pathogens (Deplancke and Gaskins, 2001). Collectedly, the combination and synergism of these immunocompetent cells have significant importance to maintaining the normal physiological functions of the intestine (Perdue and McKay, 1994; Kagnoff, 1996; MacDonald and Monteleone, 2005). Secretory IgA is the major component of the local immune barrier of the intestine and is produced locally in the intestinal mucosa, which can prevent the penetration of microorganisms, block adherence of microbes to mucus membranes, improve the bactericidal capability, and neutralize some harmful antigens across the mucosal surface (Walker, 1976; Mestecky and McGhee, 1987). Therefore, the kinetics of IgA-secreting cells in the intestine were used as an indicator to assess intestinal mucosal immunity (Zhang et al., 2007). The results of our study showed that SGAMP could increase the number of mast cells, goblet cells, and IEL from d 7 to 49 after administration of SGAMP. The data show statistically significant changes in the level of IEL, mast cells, and goblet cells in the duodenum, jejunum, and ileum of the SGAMP group, which suggests that SGAMP can improve the integrity of intestinal mucosal surface structure. Markedly increased sIgAproducing cells in the duodenum, jejunum, and ileum were also found. These results suggested that SGAMP could improve mucosal immune responses for an extended period. These results document the regulative capability of SGAMP in modulating the outcome of intestine immunocompetent cells during the whole experimental period. Further research should be conducted on the precise mechanism of SGAMP modulation of intestinal mucosal immunity, which would facilitate a better understanding of SGAMP function and exploit the potential benefits of SGAMP.

974

Wang et al. of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368–379. Schröder, J. M., and J. Harder. 1999. Human β-defensin-2. Int. J. Biochem. Cell Biol. 31:645–651. Selsted, M. E., and S. S. Harwig. 1987. Purification, primary structure, and antimicrobial activities of a guinea pig neutrophil defensin. Infect. Immun. 55:2281–2286. Selsted, M. E., S. I. Miller, A. H. Henschen, and A. J. Ouellette. 1992. Enteric defensins: Antibiotic peptide components of intestinal host defense. J. Cell Biol. 118:929–936. Selsted, M. E., and A. J. Ouellette. 1995. Defensins in granules of phagocytic and non-phagocytic cells. Trends Cell Biol. 5:114– 119. Selsted, M. E., D. Szklarek, and R. I. Lehrer. 1984. Purification and antibacterial activity of antimicrobial peptides of rabbit granulocytes. Infect. Immun. 45:150–154. Selsted, M. E., Y. Q. Tang, W. L. Morris, P. A. McGuire, M. J. Novotny, W. Smith, A. H. Henschen, and J. S. Cullor. 1993. Purification, primary structures, and antibacterial activities of β-defensins, a new family of antimicrobial peptides from bovine neutrophils. J. Biol. Chem. 268:6641–6648. Shatos, M. A., J. D. Ríos, Y. Horikawa, R. R. Hodges, E. L. Chang, C. R. Bernardino, P. A. Rubin, and D. A. Dartt. 2003. Isolation and characterization of cultured human conjunctival goblet cells. Invest. Ophthalmol. Vis. Sci. 44:2477–2486. Shi, J., C. R. Ross, M. M. Chengappa, and F. Blecha. 1994. Identification of a proline-arginine-rich antibacterial peptide from neutrophils that is analogous to PR-39, an antibacterial peptide from the small intestine. J. Leukoc. Biol. 56:807–811. Simmaco, M., G. Mignogna, D. Barra, and F. Bossa. 1994. Antimicrobial peptides from skin secretions of Rana esculenta. Molecular cloning of cDNAs encoding esculentin and brevinins and isolation of new active peptides. J. Biol. Chem. 269:11956–11961. Specian, R. D., and M. G. Oliver. 1991. Functional biology of intestinal goblet cells. Am. J. Physiol. Cell Physiol. 260:C183–C193. Steiner, H., D. Hultmark, Å. Engström, H. Bennich, and H. Boman. 1981. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292:246–248. Tani, K., W. J. Murphy, O. Chertov, R. Salcedo, C. Koh, I. Utsunomiya, S. Funakoshi, O. Asai, S. H. Herrmann, J. M. Wang, L. W. Kwak, and J. J. Oppenheim. 2000. Defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens. Int. Immunol. 12:691–700. Walker, W. A. 1976. Host defense mechanisms in the gastrointestinal tract. Pediatrics 57:901–916. Wang, D. C., J. M. Xiong, R. P. She, L. Q. Liu, Y. M. Zhang, D. M. Luo, W. G. Li, Y. X. Hu, Y. H. Wang, Q. Zhang, and Q. Sun. 2008. Mast cell mediated inflammatory response in chickens after infection with very virulent infectious bursal disease virus. Vet. Immunol. Immunopathol. 124:19–28. Xu, L. R., D. Y. Ou, and D. H. Gao. 2001. Histochemistry and morphology of mast cells of primary lymphoid organs in chickens. Chin. J. Histochem. Cytochem. 10:449–455. Yang, H., Y. Fan, R. Finaly, and D. H. Teitelbaum. 2003. Alteration of intestinal intraepithelial lymphocytes after massive small bowel resection. J. Surg. Res. 110:276–286. Yeaman, M. R., and N. Y. Yount. 2003. Mechanisms of antimicrobial peptide action and resistance. Pharmacol. Rev. 55:27–55. Zasloff, M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci. USA 84:5449–5453. Zhang, X. F., X. W. Zhang, and Q. Yang. 2007. Effect of compound mucosal immune adjuvant on mucosal and systemic immune responses in chicken orally vaccinated with attenuated Newcastledisease vaccine. Vaccine 25:3254–3262.

Downloaded from http://ps.oxfordjournals.org/ at Serials Department on October 15, 2014

Nissle 1917 on intestinal mucosal immune cells of healthy young pigs. Vet. Immunol. Immunopathol. 111:239–250. Gabay, J. E. 1994. Ubiquitous natural antibiotics. Science 264:373– 374. Ganz, T. 2003. Defensin: Antimicrobial peptides of innate immunity. Nat. Rev. Immunol. 3:710–720. Ghosh, D., E. Porter, B. Shen, S. K. Lee, D. Wilk, J. Drazba, S. P. Yadav, J. W. Crabb, T. Ganz, and C. L. Bevins. 2002. Paneth cell trypsin is the processing enzyme for human defensin-5. Nat. Immunol. 3:583–590. Guy-Grand, D., M. Dy, G. Luffau, and P. Vassalli. 1984. Gut mucosal mast cells. Origin, traffic, and differentiation. J. Exp. Med. 160:12–28. Hancock, R. E. W., and M. G. Scott. 2000. The role of antimicrobial peptides in animal defences. Proc. Natl. Acad. Sci. USA 97:8856–8861. Heller, W. T., A. J. Waring, R. I. Lehrer, and H. W. Huang. 1998. Multiple states of β-sheet peptide protegrin in lipid bilayers. Biochemistry 37:17331–17338. Kagnoff, M. F. 1996. Mucosal immunology: New frontiers. Immunol. Today 17:57–59. Korthuis, R. J., D. C. Gute, F. Blecha, and C. R. Ross. 1999. PR-39, a proline/arginine-rich antimicrobial peptide prevents postischemic microvascular dysfunction. Am. J. Physiol. Heart Circ. Physiol. 277:H1007–H1013. Lee, J. Y., A. Boman, S. Chuanxin, M. Andersson, H. Jornvall, V. Mutt, and H. G. Boman. 1989. Antibacterial peptides from pig intestine: Isolation of a mammalian cecropin. Proc. Natl. Acad. Sci. USA 86:9159–9162. Lehrer, R. I., and T. Ganz. 1990. Antimicrobial polypeptides of human neutrophils. Blood 76:2169–2181. Lehrer, R. I., and T. Ganz. 1999. Antimicrobial peptides in mammalian and insect host defence. Curr. Opin. Immunol. 11:23–27. Lehrer, R. I., M. Rosenman, S. S. Harwig, R. Jackson, and P. Eisenhauer. 1991. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods 137:167–173. Liu, T. L., R. P. She, K. Z. Wang, H. H. Bao, Y. M. Zhang, D. M. Luo, Y. X. Hu, Y. Ding, D. C. Wang, and K. S. Peng. 2008. Effects of rabbit sacculus rotundus antimicrobial peptides on the intestinal mucosal immunity in chickens. Poult. Sci. 87:250–254. Ma, W. M., R. P. She, F. Z. Peng, H. Jin, and Y. X. Hu. 2004. The preparation and patial characterization of an antimicrobial peptide from the small intestine of pig. Sci. Technol. Eng. 3:202– 205. (In Chinese). MacDonald, T. T., and G. Monteleone. 2005. Immunity, inflammation, and allergy in the gut. Science 307:1920–1925. Mestecky, J., and J. R. McGhee. 1987. Immunoglobulin A (IgA): Molecular and cellular interactions involved in IgA biosynthesis and immune response. Adv. Immunol. 40:153–245. Metcalfe, D. D., D. Baram, and Y. A. Mekori. 1997. Mast Cells. Physiol. Rev. 77:1033–1079. Morris, B. C., H. D. Danforth, D. J. Caldwell, F. W. Pierson, and A. P. McElroy. 2004. Intestinal mucosal mast cell immune response and pathogenesis of two Eimeria acervulina isolates in broiler chickens. Poult. Sci. 83:1667–1674. Nizet, V., T. Ohtake, X. Lauth, J. Trowbridge, J. Rudisill, R. A. Dorschner, V. Pestonjamasp, J. Piraino, K. Huttner, and R. L. Gallo. 2001. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414:454–457. Oppenheim, J. J., A. Biragyn, L. W. Kwak, and D. Yang. 2003. Roles of antimicrobial peptides such as defensins in innate and adaptive immunity. Ann. Rheum. Dis. 62(Suppl. 2):ii17–ii21. Perdue, M. H., and D. M. McKay. 1994. Integrative immunophysiology in the intestinal mucosa. Am. J. Physiol. Gastrointest. Liver Physiol. 267:G151–G165. Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation