- Email: [email protected]
Effects of lipopolysaccharide on the expression of proinflammatory cytokines and chemokines and the subsequent recruitment of immunocompetent cells in the oviduct of laying and molting hens
Effects of lipopolysaccharide on the expression of proinflammatory cytokines and chemokines and the subsequent recruitment of immunocompetent cells in the oviduct of laying and molting hens T. Nii, Y. Sonoda, N. Isobe, and Y. Yoshimura^ Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 139-8528, Japan ABSTRACT The goal of this study was to examine whether lipopolysaccharide (LPS) induces the expression of proinflammatory cytokines and chemokines and recruits T cells in the lower part of the oviduct, and whether that response to LPS is different between the laying and molting phase. White Leghorn laying and molting hens were intravenously injected with saline (control) or LPS. The uterus and vagina of oviducts were collected 3 or 6 h after injection, and used for reverse transcription PGR analysis of IL-lß, IL-6, IL-8 {GXGLÍ2), and lymphotactin {Lptn), and for immunohistochemical analysis for the frequency of GD4-I- and GD8-F T cells. The expressions of IL-lß, IL-6, and GXGLi2 in the uterus and that of IL-lß in the vagina were upregulated in response to LPS 3 or 6 h after injection in both laying and molting hens. The GXGLÍ2 expression in the vagina was upregulated by LPS in laying hens, whereas those effects of LPS were not significant
in molting hens. Expression of Lptn showed a tendency to be downregulated after 3 h, with recovery by 6 h after LPS injection. The frequency of GD4-I- T cells tended to increase in response to LPS after 6 h in the lamina propria of the uterus and vagina in both laying and molting hens. The GD8-I- T cell frequencies in the lamina propria of the uterus and vagina of laying hens increased in response to LPS after 6 h. However, in the molting hens, LPS stimulation resulted in GD8-f- T cell increase in the vagina only and not in the uterus. These results suggest that expressions of proinflammatory cytokines and GXGLÍ2 chemokine are upregulated in association with T cell recruitment in response to LPS in the lower part of the oviduct, although GD8+ T cells in the uterus may be depressed during the molting phase. These immunoresponses may play roles in the defense against infection of the oviduct.
Key words: chicken oviduct, molting, T cell, cytokine, chemokine 2011 Poultry Science 90:2332-2341 doi:10.3382/ps.2011-01596
INTRODUCTION The hen oviduct is susceptible to bacterial and viral pathogenic microorganisms. Infection of oviducts by these organisms (such as Salmonella organisms, Escherichia coli, and Mycoplasma) lead to functional disorders for egg formation and concomitant production of contaminated eggs (Feberwee et al., 2009; Gantois et al., 2009; Neubauer et al., 2009; Ozaki and Murase, 2009). Innate and adaptive immune systems play an essential role in the defense against infection by microorganisms. Previous studies identified macrophages, antigen-presenting cells and lymphocytes in the mucosal tissues of the oviduct (Withanage et al., 1997; Zheng et a l , 1998; Zheng and Yoshimura, 1999). Macrophages play firstdefense roles through their phagocytic activity in com©2011 Poultry Science Association Inc. Received May 10, 2011. Accepted Jnly 7, 2011. ^ Gorresponding author; [email protected]
bination with heterophils. In addition, T cells are essential for immunoresponse; GD4-I- helper T cells stimulate B cells and macrophages, whereas GD8-I- T cells exert cytotoxic effects on infected cells in response to antigens presented by major histocompatibility complex class II and I, respectively. Toll-like receptors (TLR), a member of pathogen-associated molecular pattern receptors that recognize microbial agents, are involved in the innate immune response. To date, 10 types of TLR, including TLR4, which recognizes lipopolysaccharide (LPS) of gram-negative bacteria (Takeuchi et al., 1999; Li et al., 2009), have been identified in chickens (Temperley et al., 2008). The interaction of TLR and their ligands induces cellular responses such as synthesis of proinflammatory cytokines, chemokines, and antimicrobial peptides (Kogut et al., 2006; Berndt et al., 2007; Gheeseman et a l , 2008; Abdel Mageed et al., 2011). We have reported that TLR4 is expressed in the mucosal tissue of the hen oviduct (Ozoe et al., 2009). Proinfiammatory cytokines and chemokines play significant roles in initiating innate and adaptive immu-
2332
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS
noresponses and local inflammatory responses (Staeheli et al, 2001; Ferro et al., 2004; Hughes et al, 2007). Although the primary structures of avian proinfiammatory cytokines such as IL-Iß and IL-6 are different from those of mammals, they may have similar immunoresponse tasks (Staeheli et al., 2001). The activity of ILlß includes T cell proliferation, induction of fever, triggering of acute-phase response, and activation of the vascular endothelium, although many of their activities may be mediated by other proinfiammatory cytokines and chemokines (Staeheli et al., 2001). Interleukin-6 may regulate macrophage differentiation (Chomarat et al., 2000), T cell activation (Lotz et al, 1988; Diehl et al., 2000), and immunoglobulin production by B cells (Kishimoto and Hirano, 1988). Interleukin-8 (also known as CXCLÍ2) is a chemokine responsible for an infiammatory process that may participate in the recruitment of heterophils (Poh et al., 2008; Redmond et al., 2011), monocytes (Barker et al, 1993), and CD3-f T cells (Min et al., 2001) to the site of infection in birds. Lymphotactin (Lptn) may have chemotactic effects on B cells and T cells (Rossi et al., 1999). It was shown that feed withdrawal significantly affected the immune systems in blood, which caused a higher heterophil-to-lymphocyte ratio and a decrease in the number of critical sets of T cells (Golden et al., 2008). The oviduct may have a unique regulation system for immunocompetent cell population because antigen-presenting cells and T and B cells increased with the growth of this organ during sexual maturation, probably under the effects of estrogen (Zheng et al., 1998). In the regressed oviduct of molting hens induced by feed withdrawal, the immunocompetent cell frequencies tended to decrease in the surface and subepithelial stroma of the mucosa, whereas increased in the middle part of the stroma (Yoshimura et al., 1997). Sundaresan et al. (2008) reported that expression of proinflammatory cytokines, IL-6 and GXGLi2, were upregulated in the oviduct to play roles in the regression of tissues during induced molting. However, it is unknown whether there is a difference in the induction of proinflammatory cytokine and chemokine expression and T cell recruitment in response to LPS between laying and molting hens. Holt (1993) reported that molting hens were more susceptible to Salmonella enteritidis infection. Contamination of internal egg contents by microorganisms such as Salmonella organisms occurs in the female reproductive tract (Gantois et al., 2009). Thus, it is important to know the immune activity in the regressed oviduct of molting hens to prevent the colonization of pathogens and to obtain non-contaminated eggs upon resumption of egg laying. The vagina opens to the cloaca, where various microorganisms colonize. Thus, the immunodefense system in the lower part of the oviduct (uterus and vagina) is important in preventing infection of the oviduct. The goal of this study was to examine whether LPS induces the expression of proinflammatory cyto-
2333
kines and chemokines and recruit T cells in the lower part of the oviduct, and whether that response to LPS is different between the laying and molting phase.
MATERIALS AND METHODS
Expérimentai Birds Healthy White Leghorn laying and molting hens [approximately 310-d-old hens for the analysis after 3 h of LPS or saline injection (n = 16), and 500-d-old hens for the analysis after 6 h of injections (n = 16)] were kept in individual cages under a daily light regimen of 14L:10D. The laying hens were provided with feed and water ad libitum, and were laying regularly 4 or more eggs in a clutch. Molting hens were given a regulated feed (25g/d) and free access to water, which induced cessation of egg laying after 5 to 7 d of treatment. They were used after 20 d of cessation of egg laying as the molting hen group. The laying and molting hens were intravenously injected with 100 |j,L of saline (control group; Osaka Pharmaceutical Co., Tokyo, Japan) or 1 mg of LPS (n = 4 for each treatment) at 30 min after oviposition in laying hens, and at 0900 h in molting hens, respectively. Our previous studies have shown that injection of hens with 1 mg of LPS caused elevation of immune factor expressions such as proinflammatory cytokines and ß-defensins in the oviduct (Ozoe et al, 2009; Abdel Mageed et al., 2011). Stock solution of LPS was prepared by dissolving LPS from E. coli Olli extracted by phenol (Wako Pure Chemical Industries Ltd., Osaka, Japan) in saline at a concentration of 10 mg/mL. The uterus and vagina were collected at 3 or 6 h after injection. The eggs were located in the isthmus in the 3-h group and in the uterus in the 6-h group. These samples were processed for paraffin and frozen sections and total RNA sample preparation. The handling of birds was done in accordance with the regulations of Hiroshima University for animal experiments.
Histology and Immunohistochemistry for T Cells The oviductal tissues were fixed with 10% formalin in PBS and processed for paraffin sections (4 fim) or embedded in optimal cutting temperature (OCT) compound (Saktn-a Co., Tokyo, Japan) and snap-frozen in isopentane and solid CO2 mixture to prepare frozen sections. The sections were stained with Hansen's hematoxylin and eosin. Frozen sections were air-dried on slides and fixed with acetone on ice for 30 min. Then, the sections were washed with PBS and incubated with 10% normal rabbit serum (blocking solution) for 30 min. They were incubated overnight with anti-CD4 or antiCD8 antibody (Santa Cruz Biotech. Inc., Santa Cruz, CA) dihited to 1:500 with PBS, followed by washing with PBS ( 3 x 5 min). The immunoreaction products were detected using a Histofine SAB-PO (M) kit con-
2334
NU ET AL.
taining biotinylated anti-mouse IgG -f IgA -|- IgM and streptoavidin-peroxidase (Nichirei Go., Tokyo, Japan). Briefly, the sections were incubated with biotinylated anti-mouse IgG -|- IgA -I- IgM and streptoavidin-peroxidase for 1 h each, and washed in PBS ( 3 x 5 min) after each step. Immunoprecipitates were visualized by incubating the sections with a reaction mixture of 0.02% 3,3'-diaminobenzidine tetrahydrochloride and 0.005% H2O2 in 0.05 MTris-HGl buffer (pH 7.6). The sections were counterstained with Hansen's hematoxylin.
Image Analysis for T Cell Frequencies Sections were examined under a light microscope (Nikon Eclipse E400; Nikon, Tokyo, Japan) with image analysis software (Image-Pro Plus; Media Cybernetics Inc., Silver Spring, MD). The numbers of immunopositive cells within the mucosal epithelium (3 x 10 - 1 x 10^ [ixn^) and lamina propria (1 x 10^ - 2.7 x 10^ jxm^) were counted. Then, the frequencies of the cells were calculated to be the number of cells in 1 x 10"^ |xm of a tissue. The analysis of the cell numbers was performed in triplicate on 1 section, and the average was used for the value of that tissue.
Quantitative Reverse-Transcription PCR Analysis for Expression of Proinflammatory Cytokines and Chemokines Ribonucleic acid extraction from the mucosal tissues of the uterus and vagina was performed using Sepasol RNA I Super (Nacalai Tesque Inc., Kyoto, Japan). The extracted total RNA samples were dried and dissolved in TE buffer (10 mM Tris, pH 8.0, with 1 mM EDTA). They were treated with 1 U of RQl RNaseEree DNase (Promega Go., Madison, Wl) in a PTC-100 programmable thermal controller (MJ Research Inc., Waltham, MA), programmed at 37°C for 45 min and 65°C for 10 min. The concentration of RNA in each sample was measured using Gene Quant Pro (Amersham Pharmacia Biotech, Cambridge, UK). Then, RNA samples were reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) according to the
manufacturer's instructions. The reaction mixture (10 (iL) consisted of 1 (xg of the total RNA, 1 x RT buffer, 1 mM dNTP mixture, 20 U of RNase inhibitor, 0.5 \ig of oligo(dT)2o primer, and 50 U of ReverTra Ace. The reverse transcription was performed at 42°C for 30 min, followed by heat inactivation for 5 min at 99°C using the PTC-100 programmable thermal controller (MJ Research Inc.). The real-time PCR was performed using the Roche Light Cycler system (Roche Applied Science, Indianapolis, IN). The reaction mixture consisted of 20 |iL of buffer containing 3 |iL of cDNA, 1 X SYBR Premix EX Taq (Takara, Tokyo, Japan), 0.4 \iM of each primer, and was taken into 20-|aL capillaries (Roche Diagnostics GmbH, Mannheim, Germany). Table 1 shows the primers used for PCR. The cycle parameters were PCR reaction at 95°C for 5 s and 62°C for 20 s. Eor data analysis, the A threshold cycle (CT) was calculated for each sample by subtracting the CT value of RPS17 (internal control) from the CT of the respective target gene. Eor relative quantification, the ACT value of RPS17 was then subtracted from the ACT of each experimental sample to generate the AACT. The AACT value was, therefore, fit to the formula 2"^^*-'^ to calculate the approximate fold difference. The results were expressed as fold change obtained from the ratio between the experimental samples and standard samples (corresponding oviductal tissues of non-treated hens).
Statistical Analysis The fold changes in the cytokine and chemokine expressions and frequencies of immunopositive cells were expressed as the mean ± SEM, and their significance of differences between the control and LPS groups within laying or molting hens were examined by i-test. Differences in the cytokine and chemokine expressions between laying and molting hens within control or LPS groups were also examined by i-test. However, the differences in the T cell frequency between laying and molting hens are not shown because the histological structures were not comparable between those birds. The T cells were located in the connective tissues in
Table 1. Polymerase chain reaction primers used for profiling expression of cytokines and chemokines' Target gene
Primers 5'-3'
Accession no.
IL-lb
F-GGGCATCAACCCCTACAA R-CTGTCCAGGCGGTAGAAGAT F-AGAAATCCCTCCTCGCCAAT R-AAATAGCGAACGGCCCTCA F-CTGTCCTGGCCCTCCTCCTGGTT R-TGGCGTCAGCTTCACATCTTG F-GGATTTAAGGGTGAACAGTAGATG R-TAGAAATAGAAAGCCCGAGGAT F-AAGCTGCAGGAGGAGGAGAGG R-GGTTGGACAGGCTGCCGAAGT
NM_204524
IL-6 IL-8 Lptn RPSn
= forward; R = reverse.
NM_204628.1 NM205498 AF006742 NM_204217
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS
the lamina propria. The lamina propria was filled with well-developed tubular glands and a small amount of connective tissue in laying hens, whereas it was filled mainly with connective tissue with a small number of regressed tubular glands in molting hens.
RESULTS The IL-lß expression was significantly greater in the LPS group than in the control in both uterus and vagina of laying and molting hens at 3 h after LPS injection. There was significantly higher expression of IL-lß in the LPS group than in the control only in the vagina of laying and molting hens at 6 h after injection (Figure la). The expreîssion of IL-6 was significantly higher in the LPS group than in the control in the uterus of laying hens at 3 h after injection and in the uterus of molting hens at 6 h after injection (Figure lb).
2335
The GXGLÍ2 expression was significantly higher in the LPS group than in the control in the uterus and vagina of laying hens at 3 h after injection. In molting hens, the expression was higher in the LPS group in the uterus, but not in the vagina at 3 h after injection (Figure 2a). Significant differences in GXGLÍ2 expression between the LPS and control groups were not identified at 6 h after injection in both laying and molting hens (Figure 2a). The Lptn expression showed profiles of downregulation in response to LPS in the uterus of laying and molting hens at 3 h after LPS injection, whereas the downregulation profiles were not observed at 6 h after injection (Figure 2b). The expression levels of IL-lß (Figure la), IL-6 (Figure lb), GXGLÍ2 (Figure 2a), and Lptn (Figure 2b) tended to be greater in the uterus of molting hens compared with laying hens within the control or LPS group at 3 or 6 h after injection.
z) IL-lß 160
Control
140
LPS
120 100 "O
80 60 40 20 O
M
M
uterus
vagina 3 h after injection
M
M
uterus
vagina 6 h after injection
Çb)IL-6
10 r
1
I
4
M uterus
M vagma
3 h after injection
M
M uterus
vagma
6 h after injection
Figure 1. Effects of lipopolysaccharide (LPS) on the expression of proinflammatory cytokines, IL-lß and IL-6, in the uterus and vagina of laying and molting hens. Laying (L) and molting hens (M) were injected with saline (control; D) or LPS (^) 3 or 6 h before examination. The values are the means ± SEM of fold change in the expression of IL-lß (panel a) and IL-6 (panel b), n = 4 each. Asterisks indicate values that are significantly different between the control and LPS groups (*P < 0.05 and **P < 0.01, respectively): a,b values are significantly different between laying and molting hens within control (label a) or LPS (label b) group in a corresponding segment [P < 0.05).
2336
NU ET AL.
(a) CXCLú 400 r
Control LPS
300 ~
200
II
Pl
o
100
o
M
M
uterus
M uterus
vagina
3 h after injection
M vagina
6 h after injection
(b)
30 r 25
ä
20
ce o
15 10 5 I '
O
*'^
M uterus
i*»^
M
M vagina
3 h after injection
uterus
M vagina
6 h after injection
Figure 2. Effects of lipopolysaccharide (LPS) on the expression of chemokines, IL-8 (CXCLÍ2), and lymphotactin (Lptn), in the uterus and vagina of laying and molting hens. Laying (L) and molting hens (M) were injected with saline (control; D) or LPS (0) 3 or 6 h before examination. The values are the means ± SEM of fold change in the expression of CXCLÍ2 (panel a) and Lptn (panel b), n = 4 each. Asterisks indicate values that are significantly different between the control and LPS groups {*P < 0.05 and **P < 0.01, respectively); a,b values are significantly different between laying and molting hens within control (label a) or LPS (label b) group in a corresponding segment (P < 0.05).
Histological observations showed that the mucosal epithelium of each oviductal segment was lined by a ciliated pseudostratified epithelium, and the tubular glands were developed in the lamina propria of the uterus of laying hens. In molting hens, the height of the mucosal epithelium was decreased and the tubular glands in the uterus were regressed (data not shown). Eigure 3 shows the immunolocalization of CD4-I- and CD8-t- T cells in the uterus and vagina of laying and molting hens injected with saline or LPS. The CD4-F- and CD8-F T cells were localized in the mucosal epithelium and connective tissues of the lamina propria. The frequency of CD4-I- T cells in the lamina propria of both uterus and vagina was greater in the LPS group than in the control in both laying and molting hens at 3 and 6 h after injection (Eigure 4a). There were no significant differences in the frequencies of CD4-I- T cells in the mucosal epithelium between LPS and control
groups in both segments of laying and molting hens at 3 and 6 h after injection (Eigure 4b). The CD8-I- T cell frequencies in the lamina propria of laying hens were higher in the LPS group than in the control in both uterus and vagina at 3 and 6 h after injection (Eigure 5a). However, in molting hens, significantly higher frequency was found only in the vagina at 6 h after injection, and any significant effect of LPS on CD8-I- T cell frequency was not found in the uterus (Eigure 5a). The frequency of epithelial CD8-f T cell decreased in the LPS group in the uterus of molting hens, whereas it showed no change in the uterus and vagina of laying hens and vagina of molting hens (Eigure 5b).
DISCUSSION We examined whether the expressions of proinflammatory cytokines and chemokines were induced and T
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS Laying
2337
Molting
Control
LPS
Control
LPS
F.
,
Lp
I:
1 Lp
L
F.
Lp l.p
I
r, lp
l.p
Figure 3. Micrographs showing the immunoreactive GD4-I- and GD8-I- T cells in the uterus and vagina of laying and molting hens injected with saline or lipopolysaccharide (LPS) 6 h before examination. Gompared with laying hens, the mucosal epithelium of hoth uterus and vagina is thinner and the tubular glands in the uterus are regressed in molting hens. The CD4+ and CD8-F T cells are localized in the nmcosal epithelium and connective tissues of the lamina propria in all tissues (arrows). E = mucosal epithehum; L = lumen of oviduct; Lp = lamina propria; T = tuhular gland. Scale bars = 10 nm. Color version available in the online PDF.
cells were recruited in the lower part of the oviduct (uterus and vagina) in response to LPS, and whether those responses to LPS were different between laying and molting hens. Significant findings were: (1) the expression of proinflammatory cytokines {IL-lß and IL6) and chemokines ( GXGLi2) tended to be upregulated by LPS in both laying and molting hens, and (2) the frequency of GD4+ T cells was increased by LPS in both the uterus and vagina of laying and molting hens, whereas an increase of GD8-f T cells was found in both segments in laying hens, but was only found in the vagina of molting hens. Our previous study has shown the expression of TLR4 in all oviductal segments (Ozoe et al., 2009). The current study showed higher expressions of IL-lß in the LPS group than in the control in both laying and molting hens. These results suggest that TLR4 in the uterus and vagina is able to recognize LPS in both laying and molting hens, and the interaction of LPS and TLR4 upregulated the expression of
the cytokines and chemokine in both developed and regressed oviducts. Upregulation of IL-lß expression by LPS was found in both the uterus and vagina of laying and molting hens. Significantly higher expressions of IL-6 and GXGLi2 in the LPS group were identified in the uterus of laying and molting hens at 3 or 6 h after injection. Although the GXGLÍ2 in the vagina was upregulated in laying hens, that in molting hens was not affected significantly. Thus, the response to induce GXGLÍ2 may have decreased in the vagina of molting hens. It was likely that Lptn expression was downregulated at 3 h after LPS injection, followed by recovery after 6 h in the uterus and vagina in both laying and molting hens. In avian intestinal tissues, the expressions of IL-lß, IL6, and GXGLi2 increased in response to Salmonella and CampyZo&acier bacteria (Shaughnessy et al., 2009). Salmonella Enteritidis induced GXG chemokine expression in the cecum and oviduct (Garvajal et al., 2008; Li et
2338
Nil ET AL. (a) CD4+ T cells, lamina propria
•
160 140 ,uirl o X —'
ell no.
.s O
Controi
M LPS
120 100 80
1J
60 40 20 0
M
_L
I M_
vagina
uterus
3 h after injection
M uterus
M vagina
6 h after injection
(b) CD4+ T cells, mucosai epithelium 14 12 10 8 JB
6
i
4 2
É M uterus
M vagma
3 h after injection
M uterus
M vagma
6 h after injection
Figure 4. Effects of lipopolysaccharide (LPS) on the frequency of CD44- T cells in the mucosai tissues of the uterus and vagina in laying and molting hens. Laying (L) and molting hens (M) were injected with saline (control; D) or LPS (^) 3 or 6 h before examination, (a) and (b): frequencies in the lamina propria and the mncosal epithelium, respectively. The values are the means ± SEM of the cell number in 1 x 10'^ (im^ area (n = 4 each). Asterisks indicate values that are significantly different between the control and LPS groups within a corresponding tissue (*P < 0.05 and **P < 0.01, respectively).
al., 2009). The elevated expression of proinflammatory cytokines and chemokines in response to LPS observed in this study may be the prerequisite to prevent the development of infection by gram-negative bacteria, as observed in other reports. The frequency of CD4-I- T cells became higher in the LPS group than in the control 3 h after injection in both the uterus and vagina of laying and molting hens. These results suggest that CD4-f T cells could be recruited in the lamina propria of these oviductal segments of both laying and molting hens in response to LPS. However, the recruitment of CD4-I- T cells in the mucosai epithelium may not have been enhanced because changes in their frequency were not detected. The CD8-I- T cell frequency in the lamina propria became higher in the LPS group than in the control in the
uterus and vagina of laying hens and in the vagina of molting hens 3 to 6 h after injection. However, their frequency was not affected in the lamina propria and was rather depressed in the mucosai epithelium by LPS in the uterus of molting hens. These results suggest that the ability to recruit CD8-t- T cells is confined in the vagina, but may have decreased in the uterus of molting hens. The CD4-K helper and CD8+ cytotoxic T cells that play essential roles for immunoresponse to pathogens increased in number in the ovary, oviduct, and intestinal tissues when the birds were challenged with Salmonella organisms (Withanage et al., 1998, 2003). Noujaim et al. (2008), who examined CD3-f, CD4-I-, and CD8-)- cells in the intestine of chicks, showed a higher quantity of CD8-t- T cells in hens challenged with Salmonella Enteritidis. Recent reports by Pieper
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS (a) CD8+ T cells, lamina propria
G Control
600 [N
500
Ô
400
2339
W LPS
X
C
Ö
u
300 200
I
100 0 M uterus
M vagina
M uterus
3 h after injection
1\ M
vagina
6 h after injection
(b) CD8+ T cells, mucosal epithelium
250
I
200
o
"
150
100
i
50 0
M uterus
M vagina
3 h after injection
M uterus
M vagina
6 h after injection
Figure 5. Effects of lipopolysaccharide (LPS) on the frequency of CD8-1- T cells in the mucosal tissues of the oviducts in laying and molting hens. Laying (L) and molting hens (M) were injected with saline (control; G) or LPS (^) 3 or 6 h before examination, (a) and (b): frequencies in the lamina propria and the mucosal epithelium, respectively. The values are the means ± SEM of the cell number in 1 x 10'^' |xm^ area (n = 4 each). Asterisks indicate values that are significantly different between the control and LPS groups within a corresponding tissue i^P < 0 05 and **P < 0.01, respectively).
et al. (2011) also suggested that CD8-t- -ya T cells responded to Salm.onella Typhimurium. Protection from Salmonella infection by humoral mechanism alone is unlikely because of its facultative intracellular nature (De Buck et al., 2004). The cytotoxic CD8-h T cells may play an essential role for defense against such invasive microorganisms. Thus, the lower ability to recruit CD8+ T cells in response to LPS in molting hens than in laying hens may make the uterus more susceptible to pathogenic agents during molting. The natural killer cells and T cells bear the -^6 or a ß form of the T cell receptor (Davison et al., 2008). Although the functions of TCR"fö-|- T cells have not been estabhshed., most of them in the periphery do not express either CD4 or CD8, whereas those in the gut mucosa are predominantly CD8-f (Davison et al., 2008). Analysis of the phenotypes of CD8+ T cells and their functions may
show further information on the property of oviductal mucosal immunity in future studies. Sundaresan et al. (2007, 2008) suggested that the increase in cytokine expression may play a major role in the regression of the ovary and oviduct during induced molting in chickens. The results of the current study support their observation because expression of proinflammatory cytokines and chemokines were generally higher in molting hens than in laying hens. Eurthermore, IL-lß and IL-6 may play a direct or indirect role for T cell prohferation and activation (Lotz et a l , 1988; Diehl et al., 2000; Staeheh et al., 2001), and CXCLÍ2 and Lptn may have chemotactic properties to T cells (Rossi et al., 1999; Poh et a l , 2008; Redmond et al., 2011). Therefore, the T cell frequencies in the uterus and vagina might be affected by proinfiammatory cytokines (IL-lß and IL-6) and chemokines (CXCLÍ2) ex-
2340
NII ET AL.
pressed in response to LPS. However, the reason as to why CD84- T cells were not increased by LPS in the uterus of molting hens, even though the IL-lß, IL-6, and GXGLÍ2 expressions were upregulated, remains to be explained. One of the possibilities is that some other chemokines are responsible for attracting CD8-f T cells and the function of expressing such chemoattractant factors is less active in the oviduct of molting hens. In conclusion, the current results suggest that expressions of proinflammatory cytokines and chemokine GXGLÍ2 are upregulated in association with CD4-I- and CD8-I- T cell recruitment in response to LPS in the uterus and vagina during the laying phase. Although the ability for induction of cytokines and chemokines and recruitment of CD4-t- T cells in response to LPS are retained, that for CD8-I- T cells in the uterus may be depressed during the molting phase. These immunoresponses to LPS may play roles in the defense against infection in the lower part of the oviduct, and lesser recruitment of CD8-I- T cells in the uterus may lead this tissue to be more susceptible during the molting than laying phase.
ACKNOWLEDGMENTS We thank L. Liao (Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan) for his critical reading of the manuscript. This work was supported by a Grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science (Tokyo).
REFERENCES Abdel Mageed, A. M., N. Isobe, and Y. Yoshimura. 2011. Changes in the density of immunoreactive avian ß-defensin-3 and — 11 in the hen uterus in respon.se to lipopolysaccharide inoculation. J. Poult. Sei. 48:73-77. Barker, K. A., A. Hampe, M. Y. Stoeckle, and H. Hanafusa. 1993. Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells. J. Virol. 67:3528-3533. Berndt, A., A. Wilhelm, C. Jugert, J. Pieper, K. Sachse, and U. Metlmer. 2007. Chicken cecum immune response to Salmonella entérica serovars of different levels of invasiveness. Infect. Immun. 75:5993-6007. Carvajal, B. G., U. Methner, J. Pieper, and A. Berndt. 2008. Effects of Salmonella entérica serovar Enteritidis on cellular recruitment and cytokine gene expression in caecum of vaccinated chickens. Vaccine 26:5423-5433. Cheeseman, J. H., N. A. Levy, P. Kaiser, H. S. Lillehoj, and S. J. Lamont. 2008. Salmonella Enteritidis-induced alteration of inflammatory CXCL chemokine messenger-RNA expression and histologie changes in the ceca of infected chicks. Avian Dis. 52:229-234. Chomarat, P., J. Banchereau, J. Davoust, and A. K. Palucka. 2000. IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat. Immunol. 1:510-514. Davison, F., B. Kaspers, and K. A. Schat. 2008. The avian mucosal immune system. Pages 241-271 in Avian Immunology. F. Davison, B. Kaspers, and K. A. Schat, ed. Elsevier Ltd., Amsterdam, the Netherlands. De Buck, J., F. Van Immerseel, F. Haesebrouck, and R. Ducatelle. 2004. A review: Colonization of the chicken reproductive tract
and egg contamination by Salmonella. J. Appl. Microbiol. 97:233-245. Diehl, S., J. Anguita, A. Hoffmeyer, T. Zapton, J. N. Ihle, E. Fikrig, and M. Rincón. 2000. Inhibition of Thl differentiation by IL-6 is mediated by SOCSl. Immunity 13:805-815. Feberwee, A., J. J. de Wit, and W. J. M. Landman. 2009. Induction of eggshell apex abnormalities by Mycoplasma synoviae: Field and experimental studies. Avian Pathol. 38:77-85. Ferro, P. J., C. L. Swaggerty, P. Kaiser, I. Y. Pevzner, and M. H. Kogut. 2004. Heterophils isolated from chickens resistant to extraintestinal Salmonella enteritidis infection express higher levels of proinflammatory cytokine mRNA following infection than heterophils from susceptible chickens. Epidemiol. Infect. 132:10291037. Gantois, I., R. Ducatelle, F. Pasmans, F. Haesebrouck, R. Gast, T. Humphrey, and F. Van Immerseel. 2009. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiol. Rev. 33:718-738. Golden, N. J., H. H. Marks, M. E. Coleman, C. M. Schroeder, N. E. Bauer Jr., and W. D. Schlosser. 2008. Review of induced molting by feed removal and contamination of eggs with Salmonella enierica serovar Enteritidis. Vet. Microbiol. 131:215-228. Holt, P. S. 1993. Effect of induced molting on the susceptibility of White Leghorn hens to a Salmonella enteritidis infection. Avian Dis. 37:412-417. Hughes, S., T.-Y. Poh, N. Bumstead, and P. Kaiser. 2007. Re-evaluation of the chicken MIP family of chemokines and their receptors suggests that CCL5 is the prototypic MIP family chemokine, and that different species have developed different repertoires of both the CC chemokines and their receptors. Dev. Comp. Immunol. 31:72-86. Kishimoto, T., and T. Hirano. 1988. Molecular regulation of B lymphocyte response. Annu. Rev. Immunol. 6:485-512. Kogut, M. H., C. Swaggerty, H. He, I. Pevzner, and P. Kaiser. 2006. Toll-like receptor agonists stimulate differential functional activation and cytokine and chemokine gene expression in heterophils isolated from chickens with differential innate responses. Microbes Infect. 8:1866-1874. Li, S., M. Z. Zhang, L. Yan, H. Lillehoj, L. W. Pace, and S. Zhang. 2009. Induction of CXC chemokine messenger-RNA expression in chicken oviduct epithelial cells by Salmonella entérica serovar Enteritidis via the type three secretion system-1. Avian Dis. 53:396-404. Lotz, M., F. Jirik, P. Kabouridis, C. Tsoukas, T. Hirano, T. Kishimoto, and D. A. Carson. 1988. B cell stimulating factor 2/interleukin-6 is a costimulant for human thymocytes and T lymphocytes. J. Exp. Med. 167:1253-1258. Min, W., H. S. Lillehoj, J. Burnside, K. C. Weining, P. Staeheli, and J. J. Zhu. 2001. Adjuvant effects of IL-lß, IL-2, IL-8, IL-15, IFN-a, IFN-f TGF-ß4 and lymphotactin on DNA vaccination against Eimeria acervulina. Vaccine 20:267-274. Neubauer, C , M. De Souza-Pilz, A. M. Bojesen, M. BLsgaard, and M. Hess. 2009. Tissue distribution of haemolytic Gallibacterium anatis isolates in laying birds with reproductive disorders. Avian Pathol. 38:1-7. Noujaim, J. C , R. L. Andreatti Filho, E. T. Lima, A. S. Okamoto, R. L. Amorim, and R. T. Neto. 2008. Detection of T lymphocytes in intestine of broiler chicks treated with Lactobacillus spp. and challenged with Salmonella entérica serovar Enteritidis. Poult. Sei. 87:927-933. Ozaki, H., and T. Murase. 2009. Multiple routes of entry for Escherichia eoli causing colibacillosis in commercial layer chickens. J. Vet. Med. Sei. 71:1685-1689. Ozoe, A., N. Isobe, and Y. Yoshimura. 2009. Expression of Toll-like receptors (TLRs) and TLR4 response to lipopolysaccharide in hen oviduct. Vet. Immunol. Immunopathol. 127:259 268. Pieper, J., U. Methner, and A. Berndt. 2011. Characterization of avian "(8 T-cell subsets after Salmonella enteriea serovar Typhimuriuin infection of chicks. Infect. Immun. 79:822-829. Poh, T. Y., J. Pease, J. R. Young, N. Bumstead, and P. Kaiser. 2008. Re-evaluation of chicken CXCRl determines the true gene structure: CXCLil (K60) and CXCLÍ2 (CAF/INTERLEUKIN-8) are ligands for this receptor. J. Biol. Chem. 283:16408-16415.
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS Redmond, S. B., P. Ghuamniitri, G. B. Andreasen, D. Palie, and S. J. Lamont. 2011. Proportion of circulating chicken heterophils and GXGLÍ2 expression in response to Salmonella enteritidis are affected by genetic line and inunune modulating diet. Vet. Immunol. Immunopathol. 140:323 328. Rossi, D., J. Sánchez-García, W. T. McGormack, J. F. Bazan, and A. Zlotnik. 1999. Identification of a chicken "C" chemokine related to lymphotactin. J. Leukoc. Biol. 65:87-93. Shaughnessy, R. G., K. G. Meade, S. Gahalane, B. Allan, G. Reinian, .]. J. Gallanan, and G. O'Farrelly. 2009. Innate immune gene expression differentiates the early avian intestinal response between Salmonella and Campylobacter. Vet. Immunol. Immunopathol. 132:191-198. Staeheli, P., F. Pnehler, K. Schneider, T. W. Göbel, and B. Kaspers. 2001. Gytokines of birds: Gonserved function—A largely different look. J. Interferon Gytokine Res. 21:993-1010. Sundaresan, N. R., D. Anish, K. V. H. Sastry, V. K. Saxena, J. Mohan, and K. A. Ahmed. 2007. Gytokines in reproductive remodeling of molting White Leghorn hens. J. Reprod. Immunol. 73:39-50. Sundaresan, N. R., D. Anish, K. V. H. Sastry, V. K. Saxena, K. Nagarajan, J. Subramani, M. D. Leo, N. Shit, J. Mohan, M. Saxena, and K. A. Ahmed. 2008. High doses of dietary zinc induce cytokines, chemokines, and apoptosis in reproductive tissues during regression. Gell Tissue Res. 332:543-554. Takeuchi, O., K. Hoshino, T. Kawai. H. Sanjo, H. Takada, T. Ogawa. K. Takeda, and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443-451.
2341
Temperley, N. D., S. Berhn, I. R. Paton, D. K. Griffin, and D. W. Burt. 2008. Evolution of the chicken Toll-like receptor gene family: A story of gene gain and gene loss. BMC Genomics 9:62. Withanage, G. S. K., E. Baba, K. Sasai, T. Fukata, M. Kuwamura, T. Miyamoto, and A. Arakawa. 1997. Localization and enumeration of T and B lymphocytes in the reproductive tract of laying hens. Poult. Sei. 76:671-676. Withanage, G. S. K., K. Sasai, T. Fukata, T. Miyamoto, E. Baba, and H. S. Lillehoj. 1998. T lymphocytes, B lymphocytes, and macrophages in the ovaries and oviducts of laying hens experimentally infected with Salmonella enteritidis. Vet. Imniunol. Immunopathol. 66:173-184. Withanage, G. S. K., K. Sasai, T. Fukata, T. Miyamoto, H. S. Lillehoj, and E. Baba. 2003. Increased lymphocyte subpopulations and macrophages in the ovaries and oviducts of laying hens infected with Salmonella entérica serovar Enteritidis. Avian Pathol. 32:583-590. Yoshimura, Y., T. Okamoto, and T. Tamura. 1997. Localisation of MHG class II, lymphocytes and immunoglobulins in the oviduct of laying and moulting hens. Br. Poult. Sei. 38:590-596. Zheng, W. M., and Y. Yoshimura. 1999. Localization of macrophages in the chicken oviduct: Effects of age and gonadal steroids. Poult. Sei. 78:1014-1018. Zheng, W. M., Y. Yoshimura, and T. Tamura. 1998. Effects of age and gonadal steroids on the localization of antigen-presenting cells, and T and B cells in the chicken oviduct. J. Reprod. Fértil. 114:45-54.
Copyright of Poultry Science is the property of Poultry Science Association, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
in molting hens. Expression of Lptn showed a tendency to be downregulated after 3 h, with recovery by 6 h after LPS injection. The frequency of GD4-I- T cells tended to increase in response to LPS after 6 h in the lamina propria of the uterus and vagina in both laying and molting hens. The GD8-I- T cell frequencies in the lamina propria of the uterus and vagina of laying hens increased in response to LPS after 6 h. However, in the molting hens, LPS stimulation resulted in GD8-f- T cell increase in the vagina only and not in the uterus. These results suggest that expressions of proinflammatory cytokines and GXGLÍ2 chemokine are upregulated in association with T cell recruitment in response to LPS in the lower part of the oviduct, although GD8+ T cells in the uterus may be depressed during the molting phase. These immunoresponses may play roles in the defense against infection of the oviduct.
Key words: chicken oviduct, molting, T cell, cytokine, chemokine 2011 Poultry Science 90:2332-2341 doi:10.3382/ps.2011-01596
INTRODUCTION The hen oviduct is susceptible to bacterial and viral pathogenic microorganisms. Infection of oviducts by these organisms (such as Salmonella organisms, Escherichia coli, and Mycoplasma) lead to functional disorders for egg formation and concomitant production of contaminated eggs (Feberwee et al., 2009; Gantois et al., 2009; Neubauer et al., 2009; Ozaki and Murase, 2009). Innate and adaptive immune systems play an essential role in the defense against infection by microorganisms. Previous studies identified macrophages, antigen-presenting cells and lymphocytes in the mucosal tissues of the oviduct (Withanage et al., 1997; Zheng et a l , 1998; Zheng and Yoshimura, 1999). Macrophages play firstdefense roles through their phagocytic activity in com©2011 Poultry Science Association Inc. Received May 10, 2011. Accepted Jnly 7, 2011. ^ Gorresponding author; [email protected]
bination with heterophils. In addition, T cells are essential for immunoresponse; GD4-I- helper T cells stimulate B cells and macrophages, whereas GD8-I- T cells exert cytotoxic effects on infected cells in response to antigens presented by major histocompatibility complex class II and I, respectively. Toll-like receptors (TLR), a member of pathogen-associated molecular pattern receptors that recognize microbial agents, are involved in the innate immune response. To date, 10 types of TLR, including TLR4, which recognizes lipopolysaccharide (LPS) of gram-negative bacteria (Takeuchi et al., 1999; Li et al., 2009), have been identified in chickens (Temperley et al., 2008). The interaction of TLR and their ligands induces cellular responses such as synthesis of proinflammatory cytokines, chemokines, and antimicrobial peptides (Kogut et al., 2006; Berndt et al., 2007; Gheeseman et a l , 2008; Abdel Mageed et al., 2011). We have reported that TLR4 is expressed in the mucosal tissue of the hen oviduct (Ozoe et al., 2009). Proinfiammatory cytokines and chemokines play significant roles in initiating innate and adaptive immu-
2332
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS
noresponses and local inflammatory responses (Staeheli et al, 2001; Ferro et al., 2004; Hughes et al, 2007). Although the primary structures of avian proinfiammatory cytokines such as IL-Iß and IL-6 are different from those of mammals, they may have similar immunoresponse tasks (Staeheli et al., 2001). The activity of ILlß includes T cell proliferation, induction of fever, triggering of acute-phase response, and activation of the vascular endothelium, although many of their activities may be mediated by other proinfiammatory cytokines and chemokines (Staeheli et al., 2001). Interleukin-6 may regulate macrophage differentiation (Chomarat et al., 2000), T cell activation (Lotz et al, 1988; Diehl et al., 2000), and immunoglobulin production by B cells (Kishimoto and Hirano, 1988). Interleukin-8 (also known as CXCLÍ2) is a chemokine responsible for an infiammatory process that may participate in the recruitment of heterophils (Poh et al., 2008; Redmond et al., 2011), monocytes (Barker et al, 1993), and CD3-f T cells (Min et al., 2001) to the site of infection in birds. Lymphotactin (Lptn) may have chemotactic effects on B cells and T cells (Rossi et al., 1999). It was shown that feed withdrawal significantly affected the immune systems in blood, which caused a higher heterophil-to-lymphocyte ratio and a decrease in the number of critical sets of T cells (Golden et al., 2008). The oviduct may have a unique regulation system for immunocompetent cell population because antigen-presenting cells and T and B cells increased with the growth of this organ during sexual maturation, probably under the effects of estrogen (Zheng et al., 1998). In the regressed oviduct of molting hens induced by feed withdrawal, the immunocompetent cell frequencies tended to decrease in the surface and subepithelial stroma of the mucosa, whereas increased in the middle part of the stroma (Yoshimura et al., 1997). Sundaresan et al. (2008) reported that expression of proinflammatory cytokines, IL-6 and GXGLi2, were upregulated in the oviduct to play roles in the regression of tissues during induced molting. However, it is unknown whether there is a difference in the induction of proinflammatory cytokine and chemokine expression and T cell recruitment in response to LPS between laying and molting hens. Holt (1993) reported that molting hens were more susceptible to Salmonella enteritidis infection. Contamination of internal egg contents by microorganisms such as Salmonella organisms occurs in the female reproductive tract (Gantois et al., 2009). Thus, it is important to know the immune activity in the regressed oviduct of molting hens to prevent the colonization of pathogens and to obtain non-contaminated eggs upon resumption of egg laying. The vagina opens to the cloaca, where various microorganisms colonize. Thus, the immunodefense system in the lower part of the oviduct (uterus and vagina) is important in preventing infection of the oviduct. The goal of this study was to examine whether LPS induces the expression of proinflammatory cyto-
2333
kines and chemokines and recruit T cells in the lower part of the oviduct, and whether that response to LPS is different between the laying and molting phase.
MATERIALS AND METHODS
Expérimentai Birds Healthy White Leghorn laying and molting hens [approximately 310-d-old hens for the analysis after 3 h of LPS or saline injection (n = 16), and 500-d-old hens for the analysis after 6 h of injections (n = 16)] were kept in individual cages under a daily light regimen of 14L:10D. The laying hens were provided with feed and water ad libitum, and were laying regularly 4 or more eggs in a clutch. Molting hens were given a regulated feed (25g/d) and free access to water, which induced cessation of egg laying after 5 to 7 d of treatment. They were used after 20 d of cessation of egg laying as the molting hen group. The laying and molting hens were intravenously injected with 100 |j,L of saline (control group; Osaka Pharmaceutical Co., Tokyo, Japan) or 1 mg of LPS (n = 4 for each treatment) at 30 min after oviposition in laying hens, and at 0900 h in molting hens, respectively. Our previous studies have shown that injection of hens with 1 mg of LPS caused elevation of immune factor expressions such as proinflammatory cytokines and ß-defensins in the oviduct (Ozoe et al, 2009; Abdel Mageed et al., 2011). Stock solution of LPS was prepared by dissolving LPS from E. coli Olli extracted by phenol (Wako Pure Chemical Industries Ltd., Osaka, Japan) in saline at a concentration of 10 mg/mL. The uterus and vagina were collected at 3 or 6 h after injection. The eggs were located in the isthmus in the 3-h group and in the uterus in the 6-h group. These samples were processed for paraffin and frozen sections and total RNA sample preparation. The handling of birds was done in accordance with the regulations of Hiroshima University for animal experiments.
Histology and Immunohistochemistry for T Cells The oviductal tissues were fixed with 10% formalin in PBS and processed for paraffin sections (4 fim) or embedded in optimal cutting temperature (OCT) compound (Saktn-a Co., Tokyo, Japan) and snap-frozen in isopentane and solid CO2 mixture to prepare frozen sections. The sections were stained with Hansen's hematoxylin and eosin. Frozen sections were air-dried on slides and fixed with acetone on ice for 30 min. Then, the sections were washed with PBS and incubated with 10% normal rabbit serum (blocking solution) for 30 min. They were incubated overnight with anti-CD4 or antiCD8 antibody (Santa Cruz Biotech. Inc., Santa Cruz, CA) dihited to 1:500 with PBS, followed by washing with PBS ( 3 x 5 min). The immunoreaction products were detected using a Histofine SAB-PO (M) kit con-
2334
NU ET AL.
taining biotinylated anti-mouse IgG -f IgA -|- IgM and streptoavidin-peroxidase (Nichirei Go., Tokyo, Japan). Briefly, the sections were incubated with biotinylated anti-mouse IgG -|- IgA -I- IgM and streptoavidin-peroxidase for 1 h each, and washed in PBS ( 3 x 5 min) after each step. Immunoprecipitates were visualized by incubating the sections with a reaction mixture of 0.02% 3,3'-diaminobenzidine tetrahydrochloride and 0.005% H2O2 in 0.05 MTris-HGl buffer (pH 7.6). The sections were counterstained with Hansen's hematoxylin.
Image Analysis for T Cell Frequencies Sections were examined under a light microscope (Nikon Eclipse E400; Nikon, Tokyo, Japan) with image analysis software (Image-Pro Plus; Media Cybernetics Inc., Silver Spring, MD). The numbers of immunopositive cells within the mucosal epithelium (3 x 10 - 1 x 10^ [ixn^) and lamina propria (1 x 10^ - 2.7 x 10^ jxm^) were counted. Then, the frequencies of the cells were calculated to be the number of cells in 1 x 10"^ |xm of a tissue. The analysis of the cell numbers was performed in triplicate on 1 section, and the average was used for the value of that tissue.
Quantitative Reverse-Transcription PCR Analysis for Expression of Proinflammatory Cytokines and Chemokines Ribonucleic acid extraction from the mucosal tissues of the uterus and vagina was performed using Sepasol RNA I Super (Nacalai Tesque Inc., Kyoto, Japan). The extracted total RNA samples were dried and dissolved in TE buffer (10 mM Tris, pH 8.0, with 1 mM EDTA). They were treated with 1 U of RQl RNaseEree DNase (Promega Go., Madison, Wl) in a PTC-100 programmable thermal controller (MJ Research Inc., Waltham, MA), programmed at 37°C for 45 min and 65°C for 10 min. The concentration of RNA in each sample was measured using Gene Quant Pro (Amersham Pharmacia Biotech, Cambridge, UK). Then, RNA samples were reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) according to the
manufacturer's instructions. The reaction mixture (10 (iL) consisted of 1 (xg of the total RNA, 1 x RT buffer, 1 mM dNTP mixture, 20 U of RNase inhibitor, 0.5 \ig of oligo(dT)2o primer, and 50 U of ReverTra Ace. The reverse transcription was performed at 42°C for 30 min, followed by heat inactivation for 5 min at 99°C using the PTC-100 programmable thermal controller (MJ Research Inc.). The real-time PCR was performed using the Roche Light Cycler system (Roche Applied Science, Indianapolis, IN). The reaction mixture consisted of 20 |iL of buffer containing 3 |iL of cDNA, 1 X SYBR Premix EX Taq (Takara, Tokyo, Japan), 0.4 \iM of each primer, and was taken into 20-|aL capillaries (Roche Diagnostics GmbH, Mannheim, Germany). Table 1 shows the primers used for PCR. The cycle parameters were PCR reaction at 95°C for 5 s and 62°C for 20 s. Eor data analysis, the A threshold cycle (CT) was calculated for each sample by subtracting the CT value of RPS17 (internal control) from the CT of the respective target gene. Eor relative quantification, the ACT value of RPS17 was then subtracted from the ACT of each experimental sample to generate the AACT. The AACT value was, therefore, fit to the formula 2"^^*-'^ to calculate the approximate fold difference. The results were expressed as fold change obtained from the ratio between the experimental samples and standard samples (corresponding oviductal tissues of non-treated hens).
Statistical Analysis The fold changes in the cytokine and chemokine expressions and frequencies of immunopositive cells were expressed as the mean ± SEM, and their significance of differences between the control and LPS groups within laying or molting hens were examined by i-test. Differences in the cytokine and chemokine expressions between laying and molting hens within control or LPS groups were also examined by i-test. However, the differences in the T cell frequency between laying and molting hens are not shown because the histological structures were not comparable between those birds. The T cells were located in the connective tissues in
Table 1. Polymerase chain reaction primers used for profiling expression of cytokines and chemokines' Target gene
Primers 5'-3'
Accession no.
IL-lb
F-GGGCATCAACCCCTACAA R-CTGTCCAGGCGGTAGAAGAT F-AGAAATCCCTCCTCGCCAAT R-AAATAGCGAACGGCCCTCA F-CTGTCCTGGCCCTCCTCCTGGTT R-TGGCGTCAGCTTCACATCTTG F-GGATTTAAGGGTGAACAGTAGATG R-TAGAAATAGAAAGCCCGAGGAT F-AAGCTGCAGGAGGAGGAGAGG R-GGTTGGACAGGCTGCCGAAGT
NM_204524
IL-6 IL-8 Lptn RPSn
= forward; R = reverse.
NM_204628.1 NM205498 AF006742 NM_204217
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS
the lamina propria. The lamina propria was filled with well-developed tubular glands and a small amount of connective tissue in laying hens, whereas it was filled mainly with connective tissue with a small number of regressed tubular glands in molting hens.
RESULTS The IL-lß expression was significantly greater in the LPS group than in the control in both uterus and vagina of laying and molting hens at 3 h after LPS injection. There was significantly higher expression of IL-lß in the LPS group than in the control only in the vagina of laying and molting hens at 6 h after injection (Figure la). The expreîssion of IL-6 was significantly higher in the LPS group than in the control in the uterus of laying hens at 3 h after injection and in the uterus of molting hens at 6 h after injection (Figure lb).
2335
The GXGLÍ2 expression was significantly higher in the LPS group than in the control in the uterus and vagina of laying hens at 3 h after injection. In molting hens, the expression was higher in the LPS group in the uterus, but not in the vagina at 3 h after injection (Figure 2a). Significant differences in GXGLÍ2 expression between the LPS and control groups were not identified at 6 h after injection in both laying and molting hens (Figure 2a). The Lptn expression showed profiles of downregulation in response to LPS in the uterus of laying and molting hens at 3 h after LPS injection, whereas the downregulation profiles were not observed at 6 h after injection (Figure 2b). The expression levels of IL-lß (Figure la), IL-6 (Figure lb), GXGLÍ2 (Figure 2a), and Lptn (Figure 2b) tended to be greater in the uterus of molting hens compared with laying hens within the control or LPS group at 3 or 6 h after injection.
z) IL-lß 160
Control
140
LPS
120 100 "O
80 60 40 20 O
M
M
uterus
vagina 3 h after injection
M
M
uterus
vagina 6 h after injection
Çb)IL-6
10 r
1
I
4
M uterus
M vagma
3 h after injection
M
M uterus
vagma
6 h after injection
Figure 1. Effects of lipopolysaccharide (LPS) on the expression of proinflammatory cytokines, IL-lß and IL-6, in the uterus and vagina of laying and molting hens. Laying (L) and molting hens (M) were injected with saline (control; D) or LPS (^) 3 or 6 h before examination. The values are the means ± SEM of fold change in the expression of IL-lß (panel a) and IL-6 (panel b), n = 4 each. Asterisks indicate values that are significantly different between the control and LPS groups (*P < 0.05 and **P < 0.01, respectively): a,b values are significantly different between laying and molting hens within control (label a) or LPS (label b) group in a corresponding segment [P < 0.05).
2336
NU ET AL.
(a) CXCLú 400 r
Control LPS
300 ~
200
II
Pl
o
100
o
M
M
uterus
M uterus
vagina
3 h after injection
M vagina
6 h after injection
(b)
30 r 25
ä
20
ce o
15 10 5 I '
O
*'^
M uterus
i*»^
M
M vagina
3 h after injection
uterus
M vagina
6 h after injection
Figure 2. Effects of lipopolysaccharide (LPS) on the expression of chemokines, IL-8 (CXCLÍ2), and lymphotactin (Lptn), in the uterus and vagina of laying and molting hens. Laying (L) and molting hens (M) were injected with saline (control; D) or LPS (0) 3 or 6 h before examination. The values are the means ± SEM of fold change in the expression of CXCLÍ2 (panel a) and Lptn (panel b), n = 4 each. Asterisks indicate values that are significantly different between the control and LPS groups {*P < 0.05 and **P < 0.01, respectively); a,b values are significantly different between laying and molting hens within control (label a) or LPS (label b) group in a corresponding segment (P < 0.05).
Histological observations showed that the mucosal epithelium of each oviductal segment was lined by a ciliated pseudostratified epithelium, and the tubular glands were developed in the lamina propria of the uterus of laying hens. In molting hens, the height of the mucosal epithelium was decreased and the tubular glands in the uterus were regressed (data not shown). Eigure 3 shows the immunolocalization of CD4-I- and CD8-t- T cells in the uterus and vagina of laying and molting hens injected with saline or LPS. The CD4-F- and CD8-F T cells were localized in the mucosal epithelium and connective tissues of the lamina propria. The frequency of CD4-I- T cells in the lamina propria of both uterus and vagina was greater in the LPS group than in the control in both laying and molting hens at 3 and 6 h after injection (Eigure 4a). There were no significant differences in the frequencies of CD4-I- T cells in the mucosal epithelium between LPS and control
groups in both segments of laying and molting hens at 3 and 6 h after injection (Eigure 4b). The CD8-I- T cell frequencies in the lamina propria of laying hens were higher in the LPS group than in the control in both uterus and vagina at 3 and 6 h after injection (Eigure 5a). However, in molting hens, significantly higher frequency was found only in the vagina at 6 h after injection, and any significant effect of LPS on CD8-I- T cell frequency was not found in the uterus (Eigure 5a). The frequency of epithelial CD8-f T cell decreased in the LPS group in the uterus of molting hens, whereas it showed no change in the uterus and vagina of laying hens and vagina of molting hens (Eigure 5b).
DISCUSSION We examined whether the expressions of proinflammatory cytokines and chemokines were induced and T
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS Laying
2337
Molting
Control
LPS
Control
LPS
F.
,
Lp
I:
1 Lp
L
F.
Lp l.p
I
r, lp
l.p
Figure 3. Micrographs showing the immunoreactive GD4-I- and GD8-I- T cells in the uterus and vagina of laying and molting hens injected with saline or lipopolysaccharide (LPS) 6 h before examination. Gompared with laying hens, the mucosal epithelium of hoth uterus and vagina is thinner and the tubular glands in the uterus are regressed in molting hens. The CD4+ and CD8-F T cells are localized in the nmcosal epithelium and connective tissues of the lamina propria in all tissues (arrows). E = mucosal epithehum; L = lumen of oviduct; Lp = lamina propria; T = tuhular gland. Scale bars = 10 nm. Color version available in the online PDF.
cells were recruited in the lower part of the oviduct (uterus and vagina) in response to LPS, and whether those responses to LPS were different between laying and molting hens. Significant findings were: (1) the expression of proinflammatory cytokines {IL-lß and IL6) and chemokines ( GXGLi2) tended to be upregulated by LPS in both laying and molting hens, and (2) the frequency of GD4+ T cells was increased by LPS in both the uterus and vagina of laying and molting hens, whereas an increase of GD8-f T cells was found in both segments in laying hens, but was only found in the vagina of molting hens. Our previous study has shown the expression of TLR4 in all oviductal segments (Ozoe et al., 2009). The current study showed higher expressions of IL-lß in the LPS group than in the control in both laying and molting hens. These results suggest that TLR4 in the uterus and vagina is able to recognize LPS in both laying and molting hens, and the interaction of LPS and TLR4 upregulated the expression of
the cytokines and chemokine in both developed and regressed oviducts. Upregulation of IL-lß expression by LPS was found in both the uterus and vagina of laying and molting hens. Significantly higher expressions of IL-6 and GXGLi2 in the LPS group were identified in the uterus of laying and molting hens at 3 or 6 h after injection. Although the GXGLÍ2 in the vagina was upregulated in laying hens, that in molting hens was not affected significantly. Thus, the response to induce GXGLÍ2 may have decreased in the vagina of molting hens. It was likely that Lptn expression was downregulated at 3 h after LPS injection, followed by recovery after 6 h in the uterus and vagina in both laying and molting hens. In avian intestinal tissues, the expressions of IL-lß, IL6, and GXGLi2 increased in response to Salmonella and CampyZo&acier bacteria (Shaughnessy et al., 2009). Salmonella Enteritidis induced GXG chemokine expression in the cecum and oviduct (Garvajal et al., 2008; Li et
2338
Nil ET AL. (a) CD4+ T cells, lamina propria
•
160 140 ,uirl o X —'
ell no.
.s O
Controi
M LPS
120 100 80
1J
60 40 20 0
M
_L
I M_
vagina
uterus
3 h after injection
M uterus
M vagina
6 h after injection
(b) CD4+ T cells, mucosai epithelium 14 12 10 8 JB
6
i
4 2
É M uterus
M vagma
3 h after injection
M uterus
M vagma
6 h after injection
Figure 4. Effects of lipopolysaccharide (LPS) on the frequency of CD44- T cells in the mucosai tissues of the uterus and vagina in laying and molting hens. Laying (L) and molting hens (M) were injected with saline (control; D) or LPS (^) 3 or 6 h before examination, (a) and (b): frequencies in the lamina propria and the mncosal epithelium, respectively. The values are the means ± SEM of the cell number in 1 x 10'^ (im^ area (n = 4 each). Asterisks indicate values that are significantly different between the control and LPS groups within a corresponding tissue (*P < 0.05 and **P < 0.01, respectively).
al., 2009). The elevated expression of proinflammatory cytokines and chemokines in response to LPS observed in this study may be the prerequisite to prevent the development of infection by gram-negative bacteria, as observed in other reports. The frequency of CD4-I- T cells became higher in the LPS group than in the control 3 h after injection in both the uterus and vagina of laying and molting hens. These results suggest that CD4-f T cells could be recruited in the lamina propria of these oviductal segments of both laying and molting hens in response to LPS. However, the recruitment of CD4-I- T cells in the mucosai epithelium may not have been enhanced because changes in their frequency were not detected. The CD8-I- T cell frequency in the lamina propria became higher in the LPS group than in the control in the
uterus and vagina of laying hens and in the vagina of molting hens 3 to 6 h after injection. However, their frequency was not affected in the lamina propria and was rather depressed in the mucosai epithelium by LPS in the uterus of molting hens. These results suggest that the ability to recruit CD8-t- T cells is confined in the vagina, but may have decreased in the uterus of molting hens. The CD4-K helper and CD8+ cytotoxic T cells that play essential roles for immunoresponse to pathogens increased in number in the ovary, oviduct, and intestinal tissues when the birds were challenged with Salmonella organisms (Withanage et al., 1998, 2003). Noujaim et al. (2008), who examined CD3-f, CD4-I-, and CD8-)- cells in the intestine of chicks, showed a higher quantity of CD8-t- T cells in hens challenged with Salmonella Enteritidis. Recent reports by Pieper
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS (a) CD8+ T cells, lamina propria
G Control
600 [N
500
Ô
400
2339
W LPS
X
C
Ö
u
300 200
I
100 0 M uterus
M vagina
M uterus
3 h after injection
1\ M
vagina
6 h after injection
(b) CD8+ T cells, mucosal epithelium
250
I
200
o
"
150
100
i
50 0
M uterus
M vagina
3 h after injection
M uterus
M vagina
6 h after injection
Figure 5. Effects of lipopolysaccharide (LPS) on the frequency of CD8-1- T cells in the mucosal tissues of the oviducts in laying and molting hens. Laying (L) and molting hens (M) were injected with saline (control; G) or LPS (^) 3 or 6 h before examination, (a) and (b): frequencies in the lamina propria and the mucosal epithelium, respectively. The values are the means ± SEM of the cell number in 1 x 10'^' |xm^ area (n = 4 each). Asterisks indicate values that are significantly different between the control and LPS groups within a corresponding tissue i^P < 0 05 and **P < 0.01, respectively).
et al. (2011) also suggested that CD8-t- -ya T cells responded to Salm.onella Typhimurium. Protection from Salmonella infection by humoral mechanism alone is unlikely because of its facultative intracellular nature (De Buck et al., 2004). The cytotoxic CD8-h T cells may play an essential role for defense against such invasive microorganisms. Thus, the lower ability to recruit CD8+ T cells in response to LPS in molting hens than in laying hens may make the uterus more susceptible to pathogenic agents during molting. The natural killer cells and T cells bear the -^6 or a ß form of the T cell receptor (Davison et al., 2008). Although the functions of TCR"fö-|- T cells have not been estabhshed., most of them in the periphery do not express either CD4 or CD8, whereas those in the gut mucosa are predominantly CD8-f (Davison et al., 2008). Analysis of the phenotypes of CD8+ T cells and their functions may
show further information on the property of oviductal mucosal immunity in future studies. Sundaresan et al. (2007, 2008) suggested that the increase in cytokine expression may play a major role in the regression of the ovary and oviduct during induced molting in chickens. The results of the current study support their observation because expression of proinflammatory cytokines and chemokines were generally higher in molting hens than in laying hens. Eurthermore, IL-lß and IL-6 may play a direct or indirect role for T cell prohferation and activation (Lotz et a l , 1988; Diehl et al., 2000; Staeheh et al., 2001), and CXCLÍ2 and Lptn may have chemotactic properties to T cells (Rossi et al., 1999; Poh et a l , 2008; Redmond et al., 2011). Therefore, the T cell frequencies in the uterus and vagina might be affected by proinfiammatory cytokines (IL-lß and IL-6) and chemokines (CXCLÍ2) ex-
2340
NII ET AL.
pressed in response to LPS. However, the reason as to why CD84- T cells were not increased by LPS in the uterus of molting hens, even though the IL-lß, IL-6, and GXGLÍ2 expressions were upregulated, remains to be explained. One of the possibilities is that some other chemokines are responsible for attracting CD8-f T cells and the function of expressing such chemoattractant factors is less active in the oviduct of molting hens. In conclusion, the current results suggest that expressions of proinflammatory cytokines and chemokine GXGLÍ2 are upregulated in association with CD4-I- and CD8-I- T cell recruitment in response to LPS in the uterus and vagina during the laying phase. Although the ability for induction of cytokines and chemokines and recruitment of CD4-t- T cells in response to LPS are retained, that for CD8-I- T cells in the uterus may be depressed during the molting phase. These immunoresponses to LPS may play roles in the defense against infection in the lower part of the oviduct, and lesser recruitment of CD8-I- T cells in the uterus may lead this tissue to be more susceptible during the molting than laying phase.
ACKNOWLEDGMENTS We thank L. Liao (Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan) for his critical reading of the manuscript. This work was supported by a Grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science (Tokyo).
REFERENCES Abdel Mageed, A. M., N. Isobe, and Y. Yoshimura. 2011. Changes in the density of immunoreactive avian ß-defensin-3 and — 11 in the hen uterus in respon.se to lipopolysaccharide inoculation. J. Poult. Sei. 48:73-77. Barker, K. A., A. Hampe, M. Y. Stoeckle, and H. Hanafusa. 1993. Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells. J. Virol. 67:3528-3533. Berndt, A., A. Wilhelm, C. Jugert, J. Pieper, K. Sachse, and U. Metlmer. 2007. Chicken cecum immune response to Salmonella entérica serovars of different levels of invasiveness. Infect. Immun. 75:5993-6007. Carvajal, B. G., U. Methner, J. Pieper, and A. Berndt. 2008. Effects of Salmonella entérica serovar Enteritidis on cellular recruitment and cytokine gene expression in caecum of vaccinated chickens. Vaccine 26:5423-5433. Cheeseman, J. H., N. A. Levy, P. Kaiser, H. S. Lillehoj, and S. J. Lamont. 2008. Salmonella Enteritidis-induced alteration of inflammatory CXCL chemokine messenger-RNA expression and histologie changes in the ceca of infected chicks. Avian Dis. 52:229-234. Chomarat, P., J. Banchereau, J. Davoust, and A. K. Palucka. 2000. IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat. Immunol. 1:510-514. Davison, F., B. Kaspers, and K. A. Schat. 2008. The avian mucosal immune system. Pages 241-271 in Avian Immunology. F. Davison, B. Kaspers, and K. A. Schat, ed. Elsevier Ltd., Amsterdam, the Netherlands. De Buck, J., F. Van Immerseel, F. Haesebrouck, and R. Ducatelle. 2004. A review: Colonization of the chicken reproductive tract
and egg contamination by Salmonella. J. Appl. Microbiol. 97:233-245. Diehl, S., J. Anguita, A. Hoffmeyer, T. Zapton, J. N. Ihle, E. Fikrig, and M. Rincón. 2000. Inhibition of Thl differentiation by IL-6 is mediated by SOCSl. Immunity 13:805-815. Feberwee, A., J. J. de Wit, and W. J. M. Landman. 2009. Induction of eggshell apex abnormalities by Mycoplasma synoviae: Field and experimental studies. Avian Pathol. 38:77-85. Ferro, P. J., C. L. Swaggerty, P. Kaiser, I. Y. Pevzner, and M. H. Kogut. 2004. Heterophils isolated from chickens resistant to extraintestinal Salmonella enteritidis infection express higher levels of proinflammatory cytokine mRNA following infection than heterophils from susceptible chickens. Epidemiol. Infect. 132:10291037. Gantois, I., R. Ducatelle, F. Pasmans, F. Haesebrouck, R. Gast, T. Humphrey, and F. Van Immerseel. 2009. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiol. Rev. 33:718-738. Golden, N. J., H. H. Marks, M. E. Coleman, C. M. Schroeder, N. E. Bauer Jr., and W. D. Schlosser. 2008. Review of induced molting by feed removal and contamination of eggs with Salmonella enierica serovar Enteritidis. Vet. Microbiol. 131:215-228. Holt, P. S. 1993. Effect of induced molting on the susceptibility of White Leghorn hens to a Salmonella enteritidis infection. Avian Dis. 37:412-417. Hughes, S., T.-Y. Poh, N. Bumstead, and P. Kaiser. 2007. Re-evaluation of the chicken MIP family of chemokines and their receptors suggests that CCL5 is the prototypic MIP family chemokine, and that different species have developed different repertoires of both the CC chemokines and their receptors. Dev. Comp. Immunol. 31:72-86. Kishimoto, T., and T. Hirano. 1988. Molecular regulation of B lymphocyte response. Annu. Rev. Immunol. 6:485-512. Kogut, M. H., C. Swaggerty, H. He, I. Pevzner, and P. Kaiser. 2006. Toll-like receptor agonists stimulate differential functional activation and cytokine and chemokine gene expression in heterophils isolated from chickens with differential innate responses. Microbes Infect. 8:1866-1874. Li, S., M. Z. Zhang, L. Yan, H. Lillehoj, L. W. Pace, and S. Zhang. 2009. Induction of CXC chemokine messenger-RNA expression in chicken oviduct epithelial cells by Salmonella entérica serovar Enteritidis via the type three secretion system-1. Avian Dis. 53:396-404. Lotz, M., F. Jirik, P. Kabouridis, C. Tsoukas, T. Hirano, T. Kishimoto, and D. A. Carson. 1988. B cell stimulating factor 2/interleukin-6 is a costimulant for human thymocytes and T lymphocytes. J. Exp. Med. 167:1253-1258. Min, W., H. S. Lillehoj, J. Burnside, K. C. Weining, P. Staeheli, and J. J. Zhu. 2001. Adjuvant effects of IL-lß, IL-2, IL-8, IL-15, IFN-a, IFN-f TGF-ß4 and lymphotactin on DNA vaccination against Eimeria acervulina. Vaccine 20:267-274. Neubauer, C , M. De Souza-Pilz, A. M. Bojesen, M. BLsgaard, and M. Hess. 2009. Tissue distribution of haemolytic Gallibacterium anatis isolates in laying birds with reproductive disorders. Avian Pathol. 38:1-7. Noujaim, J. C , R. L. Andreatti Filho, E. T. Lima, A. S. Okamoto, R. L. Amorim, and R. T. Neto. 2008. Detection of T lymphocytes in intestine of broiler chicks treated with Lactobacillus spp. and challenged with Salmonella entérica serovar Enteritidis. Poult. Sei. 87:927-933. Ozaki, H., and T. Murase. 2009. Multiple routes of entry for Escherichia eoli causing colibacillosis in commercial layer chickens. J. Vet. Med. Sei. 71:1685-1689. Ozoe, A., N. Isobe, and Y. Yoshimura. 2009. Expression of Toll-like receptors (TLRs) and TLR4 response to lipopolysaccharide in hen oviduct. Vet. Immunol. Immunopathol. 127:259 268. Pieper, J., U. Methner, and A. Berndt. 2011. Characterization of avian "(8 T-cell subsets after Salmonella enteriea serovar Typhimuriuin infection of chicks. Infect. Immun. 79:822-829. Poh, T. Y., J. Pease, J. R. Young, N. Bumstead, and P. Kaiser. 2008. Re-evaluation of chicken CXCRl determines the true gene structure: CXCLil (K60) and CXCLÍ2 (CAF/INTERLEUKIN-8) are ligands for this receptor. J. Biol. Chem. 283:16408-16415.
OVIDUCTAL CYTOKINES, CHEMOKINES, AND T CELLS Redmond, S. B., P. Ghuamniitri, G. B. Andreasen, D. Palie, and S. J. Lamont. 2011. Proportion of circulating chicken heterophils and GXGLÍ2 expression in response to Salmonella enteritidis are affected by genetic line and inunune modulating diet. Vet. Immunol. Immunopathol. 140:323 328. Rossi, D., J. Sánchez-García, W. T. McGormack, J. F. Bazan, and A. Zlotnik. 1999. Identification of a chicken "C" chemokine related to lymphotactin. J. Leukoc. Biol. 65:87-93. Shaughnessy, R. G., K. G. Meade, S. Gahalane, B. Allan, G. Reinian, .]. J. Gallanan, and G. O'Farrelly. 2009. Innate immune gene expression differentiates the early avian intestinal response between Salmonella and Campylobacter. Vet. Immunol. Immunopathol. 132:191-198. Staeheli, P., F. Pnehler, K. Schneider, T. W. Göbel, and B. Kaspers. 2001. Gytokines of birds: Gonserved function—A largely different look. J. Interferon Gytokine Res. 21:993-1010. Sundaresan, N. R., D. Anish, K. V. H. Sastry, V. K. Saxena, J. Mohan, and K. A. Ahmed. 2007. Gytokines in reproductive remodeling of molting White Leghorn hens. J. Reprod. Immunol. 73:39-50. Sundaresan, N. R., D. Anish, K. V. H. Sastry, V. K. Saxena, K. Nagarajan, J. Subramani, M. D. Leo, N. Shit, J. Mohan, M. Saxena, and K. A. Ahmed. 2008. High doses of dietary zinc induce cytokines, chemokines, and apoptosis in reproductive tissues during regression. Gell Tissue Res. 332:543-554. Takeuchi, O., K. Hoshino, T. Kawai. H. Sanjo, H. Takada, T. Ogawa. K. Takeda, and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443-451.
2341
Temperley, N. D., S. Berhn, I. R. Paton, D. K. Griffin, and D. W. Burt. 2008. Evolution of the chicken Toll-like receptor gene family: A story of gene gain and gene loss. BMC Genomics 9:62. Withanage, G. S. K., E. Baba, K. Sasai, T. Fukata, M. Kuwamura, T. Miyamoto, and A. Arakawa. 1997. Localization and enumeration of T and B lymphocytes in the reproductive tract of laying hens. Poult. Sei. 76:671-676. Withanage, G. S. K., K. Sasai, T. Fukata, T. Miyamoto, E. Baba, and H. S. Lillehoj. 1998. T lymphocytes, B lymphocytes, and macrophages in the ovaries and oviducts of laying hens experimentally infected with Salmonella enteritidis. Vet. Imniunol. Immunopathol. 66:173-184. Withanage, G. S. K., K. Sasai, T. Fukata, T. Miyamoto, H. S. Lillehoj, and E. Baba. 2003. Increased lymphocyte subpopulations and macrophages in the ovaries and oviducts of laying hens infected with Salmonella entérica serovar Enteritidis. Avian Pathol. 32:583-590. Yoshimura, Y., T. Okamoto, and T. Tamura. 1997. Localisation of MHG class II, lymphocytes and immunoglobulins in the oviduct of laying and moulting hens. Br. Poult. Sei. 38:590-596. Zheng, W. M., and Y. Yoshimura. 1999. Localization of macrophages in the chicken oviduct: Effects of age and gonadal steroids. Poult. Sei. 78:1014-1018. Zheng, W. M., Y. Yoshimura, and T. Tamura. 1998. Effects of age and gonadal steroids on the localization of antigen-presenting cells, and T and B cells in the chicken oviduct. J. Reprod. Fértil. 114:45-54.
Copyright of Poultry Science is the property of Poultry Science Association, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.