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Poult Sci 2007. 86:1351-1355
© 2007 Poultry Science Association
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IMMUNOLOGY, HEALTH, AND DISEASE: Research Note

Ontogeny of Cytokine Gene Expression in the Chicken Spleen

M. F. Abdul-Careem, D. B. Hunter, M. D. Lambourne, J. Barta and S. Sharif1

Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada

1 Corresponding author: shayan{at}uoguelph.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytokine gene expression in the chicken spleen during embryogenesis and the early posthatch period was investigated in the present study. The constitutive expression of interleukin-4, interleukin-10, interleukin-18, and interferon-{gamma} genes was detectable as early as embryonic day 12. Expression of cytokine genes was higher in the spleen of posthatch chickens compared with chick embryos. There was a gradual increase in expression of all the cytokine genes in the spleen, which peaked by d 7 posthatch. This expression pattern coincided with the completion of T-cell colonization and structural development of the spleen during the early posthatch period. It is therefore possible that the cytokines examined in the present study are involved in the maturation of colonized T cells and in shaping the spleen microenvironment.

Key Words: cytokine • gene expression • embryo • spleen • chicken


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The spleen is a secondary lymphoid organ that plays a major role in elicitating the immune response against systemically acquired antigens (Jeurissen, 1991). The spleen microenvironment, which consists of periarteriolar and periellipsoid lymphoid sheaths, is necessary for elicitation of the antibody-mediated immune response against thymus-dependent antigens. Colonization of the peripheral lymphoid organs, including the spleen, takes place in 3 distinct waves of T cells derived from the thymus during embryonic and early posthatch stages of the chicken (Dunon et al., 1997, 1998). This occurs subsequent to the colonization of the thymus with hematopoietic precursors (Dunon et al., 1997, 1998; Siatskas and Boyd, 2000). The first, second, and third waves of T cells reach the chicken spleen between embryonic day (ED)15 and ED20, ED21 and d (D)6 posthatch, and D7 and D9, respectively. The acquisition of T cells by the developing spleen increases in magnitude, with lower numbers acquired during embryogenesis and highest numbers acquired during the early posthatch period (Dunon et al., 1998). Lowenthal and coworkers (1994) studied T-cell populations in the spleen during the early posthatch period and found that spleen CD3+ T cells were increased 18- and 73-fold on D4 and D7, respectively, when compared with D1. This increase in T cell numbers is accompanied by 4.5- and 8-fold increases in CD4+ T cells on D4 and D7, respectively, compared with D1. During the same time period, CD8+ T cells are also increased in the developing spleen. Mast and Goddeeris (1999) observed the microenvironment of the spleen during embryo development and the early posthatch period. Histologically, the typical spleen structure first appears on ED20, and by D7 the structure resembles the adult spleen architecture (Jeurissen, 1991). All these changes are associated with a gradual increase in spleen weight in both embryos and early posthatch chickens (Seto 1981).

Little information is available on the acquisition of spleen functional ability during embryogenesis and the early posthatch period. Mast and Goddeeris (1999) studied the response of embryos and early posthatch chickens (ED16, ED18, D1, D7, and D12) immunized with a thymus-dependent antigen and found that only the D12 chickens were capable of mounting significant IgG and IgM responses. Lehtonen and coworkers (1989) studied the ontogeny of alloreactivity in chickens using a mixed-lymphocyte reaction with spleen cells and recorded that spleen cells from D7 chickens had higher alloreactivity compared with D3 chickens, whereas D1 chickens lacked the capacity for alloreactivity. Seto (1989) compared the immune responsiveness of ED19 embryos and chickens between D2 and D9 and discovered that D8 chickens could mount higher immune responses in the spleen compared with the younger chickens and embryos. The increase in T cell populations in the spleen from D1 to D7 chickens has also been associated with an increase in interferon (IFN)-{gamma} in the culture supernatant of splenocytes stimulated with mitogens (Lowenthal et al., 1994). All these studies provide evidence that the functional ability of the spleen as a secondary lymphoid organ increases during embryogenesis and becomes fully competent by D7 to D8. This trend in age-dependent maturation of the immune system has also been demonstrated in mammals (Gasparoni et al., 2003; Brown et al., 2006).

The thymus, which is a primary lymphoid organ, is capable of producing cytokines in the absence of any stimulation (Yarilin and Belyakov, 2004). The constitutive expression of cytokine genes, such as interleukin (IL)-7, IL-9, IL-13, and IL-15 during T-cell development in the mouse fetal intestine and thymus has been described (Murray et al., 1998). Expression of an additional set of cytokine genes, including IL-1{alpha}, IL-2, IL-3, IL-4, IL-5, IL- 6, IFN-{gamma}, granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-{alpha}, during thymus development in the mouse has also been described (Montgomery and Dallman, 1997). On ED14, ED18, and D1, chicken cytokine genes, such as type 1 IFN, IL-1ß, IL-2, and transforming growth factor-ß4, are constitutively expressed in thymocytes, and they appear to be associated with the development of the thymus (Peters et al., 2003). In the thymus, constitutive cytokine expression is associated with the colonization and development of thymocytes (Yarilin and Belyakov, 2004). Similar to the thymus, the spleen also undergoes colonization of T cells followed by structural development and maturation of the tissue. However, the involvement of cytokines in the development of the spleen has not been studied.

The objective of the present study was to elucidate the molecular ontogeny of the chicken immune system. To address this objective, we used quantitative real-time PCR assays to determine the expression of selected cytokine genes including T helper (Th)1 (IL-18 and IFN-{gamma}) and Th2 (IL-4 and IL-10) in the spleen during embryonic development and the early posthatch period to demonstrate that the expression of these genes is developmentally regulated and may indicate the acquisition of functional maturation during the ontogeny of the spleen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Embryos and Chickens
Eggs of the Barred Rock strain of chickens were obtained and hatched at the Arkell Poultry Research Unit, University of Guelph. Chicks were housed in an isolation facility (Ontario Veterinary College, University of Guelph). Spleens of 6 embryos and early posthatch chickens were collected at each of the following time points following euthanasia by freezing or carbon dioxide inhalation, respectively: ED12, ED14, ED16, ED18, and ED20, or D1, D4, D7, and D10. Tissues were preserved in RNA-later (catalog no. 76106, Qiagen Inc., Mississauga, Ontario, Canada). On D4, only 5 chickens were sampled.

RNA Extraction and Reverse Transcription
Extraction of RNA was carried out using Trizol (catalog no. 15596-018, Invitrogen Canada Inc., Burlington, Ontario, Canada) as has been described previously (Abdul-Careem et al., 2007). Reverse transcription (RT) of total RNA (2 µg except on ED12, which was 1 µg) was carried out using Oligo(dT)12–18 primers (catalog no. 12371-019, Superscript First-Strand Synthesis System, Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.

Primers
Previously published primers were used for the relative quantification of target gene expression (IL-4, IL-10, IL-18, and IFN-{gamma}), and ß-actin was used as the reference gene (Abdul-Careem et al., 2006, 2007). The primers were synthesized by Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada).

Preparation of Constructs as Standards
Reverse-transcription PCR quantification of cytokine gene expression was done using standard curves. The standard curves for IL-4, IL-10, IL-18, IFN-{gamma}, and ß-actin have been described previously (Abdul-Careem et al., 2006, 2007).

Real-Time RT-PCR
Each real-time RT-PCR assay was run along with a dilution series of the standard that served as the calibrator. A no-template control was also included with each run. All the real-time RT-PCR runs were conducted in glass capillaries (catalog number 11909 339 001, Roche Diagnostics GmbH, Mannheim, Germany) in a final volume of 20 µL of LightCycler FastStart DNA Master SYBR Green 1 (catalog number 12 239 264 001, Roche Diagnostics GmbH) containing fast-start Taq DNA polymerase for "hot start" and DNA intercalated dye SYBR Green 1 dye for detection in a LightCycler instrument, version 3.5 (catalog number 2011 468, Roche Diagnostics GmbH). In addition, the reaction consisted of 0.25 µM each gene- specific primer, 3 mM MgCl2 (5 mM MgCl2 used for IL-4), and PCR-grade water.

The optimum thermal-cycling parameters varied according to the gene and included 1) preincubation at 95°C for 10 min; 2) 40 cycles (60 cycles used for IL-4 and IFN-{gamma}) of amplification at 95°C for 10 s (segment 1), 64°C for 5 s (segment 2), and 72°C for 10 s except for IL-10, which required only 5 s (segment 3); 3) a melting curve analysis at 95°C for 0 s (segment 1), 65°C/15 s (segment 2), and 95°C/0 s (segment 3); and 4) cooling at 40°C/30 s. Fluorescent acquisition was done between 80 and 84°C for 3 s depending on the melting temperature of the PCR product of the target or reference gene.

Data Analysis
Quantification of cytokine gene expression by RT-PCR was done as detailed in Abdul-Careem et al. (2006). Expression of cytokine genes was calculated relative to expression of the ß-actin gene and was expressed as a ratio. Expression of the target gene observed at each time point was compared with expression of the same gene on ED12, and fold changes were calculated. The fold changes in relative gene expression were subjected to 1-way ANOVA using the statistical package Minitab, release 14 (Minitab Inc., State College, PA) to identify significant differences over time. Results from the analyses were then used in a Tukey’s pairwise comparison to identify differences. Comparisons were considered significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of IL-18 and IFN-{gamma} Genes in the Spleen During Embryonic Development and Early Posthatch Periods
Expression of the IL-18 gene in the spleen was detectable as early as ED12, and expression continued at all other time points during the embryonic and early post-hatch periods (Figure 1AGo). Interleukin-18 gene expression in the spleen was significantly higher on D7 when compared with the embryonic time points (P ≤ 0.001). Similarly, IL-18 gene expression on D10 was significantly higher (P≤ 0.001) than the level of expression in embryos except for ED16. In general, IL-18 gene expression in the spleen was 3.2- to 5.4-fold higher at posthatch time points compared with the level of expression at ED12, whereas this ratio was <2-fold at all embryonic time points.


Figure 1
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  		Figure 1. Cytokine gene expression in spleen during embryogenesis and early posthatch development. (A) Interleukin (IL)-18, (B) interferon (IFN)- {gamma}, (C) IL-10, and (D) IL-4. ED12, ED14, ED16, ED18, and ED20 = age of the embryo in days; D1, D4, D7, and D10 = age of the chicken in days posthatch. Ribonucleic acid was extracted from 5 to 6 spleens from embryos or chickens at each time point and reverse transcribed, and gene expression was quantified by real-time PCR using SYBR Green (Roche Diagnostics GmbH, Mannheim, Germany). T helper (Th)1 and Th2 cytokine gene expression was calculated relative to ß-actin expression, normalized to a calibrator, and is presented as the fold change compared with expression of the same gene on ED12. Error bars represent SEM. The differences in cytokine expression over time were assessed by ANOVA followed by Tukey’s test, and comparisons were considered significant at P ≤ 0.05. a = significant when compared with observations made during embryonic stages; b = significant when compared with observations made during embryonic stages except ED16; c = significant when compared with all time points except ED16 and D10; d = significant when compared with all time points.

 
The pattern of IFN-{gamma} expression in the spleen during embryonic development and the early posthatch period was similar to that of IL-18 gene expression, being quantifiable at all time points and higher during the early post-hatch period (2.8- to 9.6-fold) compared with the level of gene expression during embryonic development, which was always <2-fold relative to ED12 (Figure 1BGo). Interferon-{gamma} gene expression in the spleen was significantly higher only on D7 when compared with that during embryo development (P ≤ 0.05).

Expression of IL-4 and IL-10 Genes in the Spleen During Embryonic Development and Early Posthatch Periods
Similar to expression of the IL-18 and IFN-{gamma} genes in the spleen, expression of the IL-10 gene was detectable on ED12 and then at all other time points during the pre-and posthatch periods (Figure 1CGo). Expression of the IL-10 gene fluctuated during embryo development and the early posthatch period, with notable peaks on ED16 (10.7 ± 9.1-fold), D7 (35.0 ± 51.1-fold), and D10 (9.9 ± 11.7). However, only on D7 was the expression of IL-10 significantly higher than the level of expression of this gene at different embryonic time points (P ≤ 0.05). There was also no significant difference between IL-10 expression at D7 and expression at ED16 and D10.

Interleukin-4 mRNA was detectable in the spleen at all time points during the pre- and posthatch periods, but only on D7 was IL-4 gene expression (8.4 ± 11.2) significantly higher (P ≤ 0.05) compared with gene expression at all other pre- and posthatch time points (Figure 1DGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study examined acquisition of constitutive expression of the IL-4, IL-10, IL-18, and IFN-{gamma} cytokine genes in the spleen during development of the chick embryo and in early posthatch chickens using quantitative real-time RT-PCR. The main findings of our study were that cytokine gene expression was significantly lower in the spleen of chick embryos compared with posthatch chicks, particularly D7 chicks. Significant expression of these cytokine genes in the spleen of D7 chickens may be an indicator of acquisition of the functional ability of the spleen as a secondary lymphoid organ.

In the present study, observations were made beginning at ED12 and extending up to D10. This was based on the fact that the spleen and other secondary lymphoid organs are populated in 3 waves, starting from ED15 to ED20, through D1 to D5, to D7 to D9 (Dunon et al., 1998, 1997; Siatskas and Boyd, 2000). We discovered that the IL-4, IL-10, IL-18, and IFN-{gamma} genes were constitutively expressed in the spleen during embryonic development, starting from ED12 and peaking by D7. The results of the present study provide evidence that the expression pattern of these cytokine genes may coincide with the completion of colonization of the spleen with T lymphocytes during the early posthatch period (Dunon et al., 1998, 1997; Siatskas and Boyd, 2000). The pattern of cytokine gene expression observed in the present study also parallels the observations made by Bar-Shira and coworkers (2003) in another avian secondary lymphoid tissue, the gut-associated lymphoid tissue (GALT). In the early posthatch period, avian GALT also shows constitutive expression of IL-2 and IFN-{gamma} and maturation of GALT; these cytokines appear to be biphasic during the post-hatch period, with one phase around D4 and the next one around D8.

Consistent with the completion of spleen colonization observed previously (Dunon et al., 1997) and cytokine gene expression observed in this study, Seto (1988) showed that the reactivity of chicken spleen to antigens is higher in magnitude in D7 and D14 chickens when compared with embryos and D2 chickens. Further, the antibody-mediated immune responses to thymus-dependent antigens, which require priming in the spleen microenvironment, are significantly higher when D12 chicks are immunized compared with antibody responses elicited in chick embryos (ED16, ED18) or young chicks (D1, D7; Mast and Goddeeris, 1999). Zhang and Sharma (2003) exposed embryos at ED4, ED8, ED10, ED12, ED14, and ED18 as well as D1 chicks to viral antigens and showed that virus exposure was only tolerogenic in embryos younger than ED14. This study concluded that ED18 embryos are mature enough to mount an immune response. This conclusion is further strengthened by the fact that administration of vaccines on d 18 of embryonation confers immunity against Marek’s disease, and it is as effective as posthatch vaccination (Sharma and Burmester, 1982). This is in contrast to our observation that the level of cytokine gene expression in the spleen is still relatively low by ED18. However, it is possible that although the constitutive levels of cytokine expression are low at ED18, antigen stimulation may result in the rapid induction of cytokine gene expression in lymphoid tissues of chick embryos. The low level of cytokine expression in spleens of early posthatch chicks may have implications for immunity against infections. For example, chickens during the early posthatch period are more susceptible to bacterial infections, such as Salmonella enterica serovar Typhimurium infection, and they mount lower immune responses to bacterial antigens compared with adult chickens (Shivaprasad, 2003; Beal et al., 2004). More studies are required to examine the possible influence of low cytokine expression and susceptibility to infections in early posthatch chickens.

The constitutive expression of both Th1 (IL-18 and IFN-{gamma}) and Th2 (IL-4 and IL-10) cytokine genes suggests that naíve chicken Th cells may express cytokine genes before being activated under Th1- or Th2-polarizing conditions. However, T-cell colonization of the spleen begins by ED15 and the source of cytokines in ED12 and ED14 chickens could therefore be other cells, including natural killer cells. Göbel and coworkers (1994) showed that natural killer cells are present in the spleen by ED8 and that their number peaks by ED14. Alternatively, cytokines may be expressed by cells of the reticuloendothelial system (RES), because these cells are known to be present from ED10 onward (Jeurissen and Janse, 1989). Seto (1988) reported the elicitation of spleen enlargement in embryonic and neonatal periods (ED14, ED18, D2, D7, and D14) after immunization with mouse red blood cells and attributed these changes to the reactions mounted by cells of the RES. Macrophages are known to be among the cells that constitute the RES. Therefore, it is possible that some of the cytokines, particularly IL-18 and IL-10, are expressed by macrophages in embryonic spleens.

In conclusion, the present study reports the developmental patterns of expression of IL-4, IL-10, IL-18, and IFN-{gamma} cytokine genes in the chicken spleen. The constitutive expression of these cytokines in the spleen as early as ED12 suggests that they may be associated with shaping the spleen environment. In addition, the expression patterns of these cytokines coincide with the completion of colonization of the spleen by recent cellular emigrants from the thymus. Further studies will be required to identify the cellular source of the cytokines and their function in mediating immune response in the embryonic and early posthatch periods.


   ACKNOWLEDGMENTS
 
M. F. Abdul-Careem is a recipient of a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC) and postgraduate scholarship supplement from the Canadian Poultry Research Council (CPRC). This study was funded by NSERC, the Poultry Industry Council, the Ontario Ministry of Agriculture, Food and Rural Affairs, and the Saskatchewan Chicken Industry Development Fund. We would like to acknowledge the staff of the animal isolation facility and Arkell Poultry Research Station of the University of Guelph for raising the chickens and incubating the eggs, respectively. Pete Kaiser (Institute of Animal Health, Berkshire, UK) is acknowledged for the chicken IL-4 plasmid construct.

Received for publication January 4, 2007. Accepted for publication March 17, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abdul-Careem, M. F., B. D. Hunter, P. Parvizi, H. R. Haghighi, N. Thanthrige-Don, and S. Sharif. 2007. Cytokine gene expression patterns associated with immunization against Marek’s disease in chickens. Vaccine 25:424–432.[ISI][Medline]

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Bar-Shira, E., D. Sklan, and A. Friedman. 2003. Establishment of immune competence in the avian GALT during the immediate post-hatch period. Dev. Comp. Immunol. 27:147–157.[ISI][Medline]

Beal, R. K., P. Wigley, C. Powers, S. D. Hulme, P. A. Barrow, and A. L. Smith. 2004. Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge. Vet. Immunol. Immunopathol. 100:151–164.[ISI][Medline]

Brown, D. C., C. V. Maxwell, G. F. Erf, M. E. Davis, S. Singh, and Z. B. Johnson. 2006. Ontogeny of T lymphocytes and intestinal morphological characteristics in neonatal pigs at different ages in the postnatal period. J. Anim. Sci. 84:567–578.[Abstract/Free Full Text]

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